EP1899702A2

EP1899702A2 - Device and method for analyzing a sample liquid

Info

Publication number: EP1899702A2
Application number: EP06742544A
Authority: EP
Inventors: Ilse Ballhorn; Gert Blankenstein; Ralf-Peter Peters; Birgit MÜLLER-CHORUS; Michael Schlüter
Original assignee: Boehringer Ingelheim Microparts GmbH
Current assignee: Boehringer Ingelheim Microparts GmbH
Priority date: 2005-04-09
Filing date: 2006-04-07
Publication date: 2008-03-19
Also published as: US20070189927A1; JP4837725B2; WO2006108559A3; JP2008534972A; WO2006108559A2; US7731907B2

Abstract

Disclosed are a device and a method for analyzing a sample liquid, particularly the ELISA method being very easy, fast, and highly accurate to carry out. According to the invention, a sample liquid and a diluting liquid are fed to several dosing chambers having different volumes such that the sample liquid can be diluted into associated reaction chambers at different dilution ratios in a dilution step. Different liquids can be delivered successively to the reaction chambers by means of a common receiving chamber. The liquids are transferred from the reaction chambers into associated test chambers in order to stop the detection reaction.

Description

Device and method for examining a sample liquid

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

The present invention is concerned with microfluidic systems or devices whose structures have a size of about 1 to 1000 μm and / or whose cavities each have a volume of about 1 to 1000 μl. The following statements relate in particular to devices and methods in which capillary, pressure and / or centrifugal forces act and are crucial in particular for the function.

The term "ELISA" comes from the English language and means "enzyme-linked immunosorbent assay". In the context of the present invention, this term is to be understood as meaning a process in which an enzyme is bound to an analyte, in particular to a complex of an analyte and an antibody. By means of the enzyme, a substrate is modified or converted into a detection substrate, in particular a fluorescent substance or the like, in a detection reaction. By detecting the detection substrate, a quantitative determination of the analyte in the sample liquid is possible. In order to enable a high accuracy and a corresponding measuring range, a dilution series of the sample liquid is usually examined in this way.

So far, the ELISA method is usually performed manually or automatically - for example by means of pipetting robots - on an open pipetting plate with, for example, 96 open Aufhahmekammern. A sample liquid to be examined is diluted several times in succession in the receiving chambers in order to achieve different dilution ratios. Subsequently, the sample liquid with the different dilution ratios is pipetted into prepared Aumahmekammern in which an analyte in the sample liquid can be bound to immobilized antibodies. After a relatively long reaction time, multiple rinsing with a washing liquid takes place. Then one is sent to a add antibody bound enzyme. The detection antibody binds to the complex of analyte and immobilized antibody. Then again different washing steps are required. Then, a substrate is added, which is converted or modified by the enzyme into a detection substrate. This detection reaction is very time critical. The stopping of the detection reaction takes place, for example, by adding acid. The problem is that this can not be done simultaneously in all Aufhahmekammern in which the detection reaction takes place, and that at higher volumes by diffusion and / or mixing processes different delays may occur. Finally, the detection substrate is determined, for example, optically, in particular by fluorescence measurement or the like. From the values determined, the concentration of the analyte in the sample liquid can be determined. The described method is very complicated and error-prone. In particular, inaccuracies add up due to the large number of individual steps. Furthermore, the preparation of the receiving chambers for immobilizing the antibody is correspondingly expensive and also associated with the use of large amounts of liquid. In addition, due to the large quantities of liquid and correspondingly large diffusion paths, the reactions often proceed very slowly, so that the ELISA method in the hitherto customary form is very time-consuming.

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 microfluid - Dical system in the form of a so-called compact disk (CD) for individual steps of the ELI S A method. There are a sample liquid, a washing liquid, a liquid filled with a detection antibody and a substrate liquid in corresponding Aumahmekammern, which are successively passed through correspondingly different rotation of the CD in a single associated reaction chamber for the corresponding reaction. Thus, individual steps in the microfluidic system can be performed. However, the Pipettieraufwand not reduced significantly, because compared to the conventional ELIS A method only the respective multiple washing steps could be avoided. In general, a large number of microfluidic systems in the form of CDs are already known in which a control of liquid flows takes place by rotation of the CD, that is to say by centrifugal forces.

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 known from US 6,706,519 Bl, US 6,719,682 B2, US 2004/0203136 A1, WO 00/78455 A1 and WO 01/87485 A2.

US 2004/0203136 A1 discloses a method and an apparatus for the examination and dilution of samples and reaction liquids. Several dosing channels are connected via a common channel to a first receiving chamber for a sample and can be filled with the sample. Furthermore, a second receiving chamber for a dilution liquid is connected to the common channel and thus to the metering channels. With a correspondingly high degree of rotation, the dilution liquid is conducted via the common channel into the metering channels, so that the metered sample quantities are transferred into subsequent mixing chambers, which are finally completely filled by the inflowing dilution liquid. This does not allow optimal or universal dilution.

The present invention has for its object to provide an apparatus and a method for examining a sample liquid, wherein a cost-effective, rapid and / or accurate quantitative examination, in particular by means of the ELISA method, is made possible.

The above object is achieved by a device according to claim 1 or 12 or by a method according to claim 18 or 25. Advantageous developments are the subject of the dependent claims.

One aspect of the present invention resides in a plurality of first metering chambers for preferably exclusively receiving sample liquid - A -

a first, common Aufhahmekammer and a plurality of second metering chambers for preferably exclusive inclusion of a dilution liquid from a second, common Aumahmekammer provide. The first and / or second metering chambers vary in their volumes. The first and second metering chambers are pairwise associated with each other and each associated with an associated reaction chamber, so that by pressure and / or centrifugal forces contained in the first and second metering chambers volumes of sample liquid and dilution liquid into the respective associated reaction chambers are transferable and miscible, whereby the sample liquid is diluted with different dilution ratios. This proposed dilution is also referred to below as the "parallel dilution". Thus, with minimal pipetting effort - it is only necessary to fill the first and second common receiving chamber with liquids from the outside - a dilution series of the sample liquid can be realized with very high accuracy.

In particular, in the proposed dilution, the inaccuracies or errors arising in the prior art, such as US 2004/0203136 A1, by using common channels or the like, are avoided. The metering of the first and second fluid is carried out independently of each other, so that otherwise occurring subsequent errors in the dosage can be avoided. Further, the first and second metering chambers are preferably connected via separate channels to the first and second Aumahmekammer so that no undefined premixtures, impurities or mixing errors can occur.

Another advantage over the prior art, such as US 2004/0203136 Al, lies in the fact that the two liquids are mixed only in the respective reaction chamber - ie quickly or selectively and / or under defined conditions, so that, for example fast reactions can take place in a defined manner. In particular, the liquids from the first and second metering chambers can be transferred and mixed simultaneously or successively into the reaction chambers. Particularly preferably, the volumes of the first and second metering chambers vary in opposite directions. If the metering chambers are arranged, for example, in two rows running parallel to each other or parallel, the volume of the first metering chambers increases in one direction (in particular optionally in or opposite to the filling direction), while the volume of the second metering chambers decreases in this direction. Thus, with a small space requirement and with small liquid volumes, a dilution series can be realized over a large dilution range.

Preferably, the individual sums of the mating pairs of first and second metering chambers are the same. This is an optimal space utilization, especially on a CD, conducive. Furthermore, equal volumes of diluted sample liquid with different dilution ratios result, so that correspondingly the other subsequent cavities, in particular reaction chambers and the like, can all be uniformly designed for the same volumes, whereby the design is simplified and standardized.

According to a preferred embodiment, a single parallel dilution suffices to cover a sufficiently large dilution range. However, if required, at least one further, preferably also parallel, dilution can take place after the first parallel dilution. This sub-dilution can be done, for example, only for one, for example, the already diluted with the largest dilution ratio Probenflüssig- keitsmenge. However, if necessary, several or all of the liquid volumes of differently diluted sample liquid produced by the first parallel dilution may each be subjected to a separate further, in particular likewise parallel, dilution.

The dilution liquid already used or supplied for the first dilution is preferably used for the further dilution. Then a renewed supply of dilution liquid is not required, whereby the handling simplifies, in particular the required pipetting of liquids is minimized. According to a further, independently realizable aspect of the present invention, a (third) common Aumahmekanimer is provided for a plurality of reaction chambers. In particular, several liquids can be supplied one after the other in the receiving chamber-that is to say sequentially-for example by pipetting or in some other way-in particular, therefore, externally or externally. Thus, a common Einfüllöffhung for particular different liquids created and usable. An undesired mixing of the various liquids in the receiving chamber and a sequential transfer into the reaction chambers, which are preferably connected in parallel, are made possible in that the receiving chamber is emptied before receiving a new liquid, in particular automatically by capillary forces and / or by centrifugal forces.

In particular, it becomes possible to prepare several or all reaction chambers in a suitable manner with minimal effort, in particular with very few pipetting operations, ie, for example, to immobilize a reagent, such as an antibody or the like, in the reaction chambers. Alternatively or additionally, the common receiving chamber assigned to the reaction chambers allows the detection reaction to be carried out, for example by feeding corresponding liquids with the enzyme, the substrate or the like, with minimal pipetting effort.

A further aspect of the present invention resides in the fact that the reaction chambers are each assigned a detection or examination chamber and the detection reactions which preferably take place enzymatically by an immobilized enzyme in the reaction chambers can be stopped by the liquid present in the reaction chambers being introduced into the associated examination chamber - Preferably by pressure, capillary and / or centrifugal forces - is transferred. This transfer takes place in particular simultaneously for several or all reaction chambers, so that the detection reactions can also be stopped simultaneously in these reaction chambers. The examination, in particular the detection of detection substrate or the like formed in the respective liquid, can then take place successively in the examination chambers as required. Thus, a considerably greater accuracy is achieved when stopping the particular enzymatic table and accordingly time-critical detection reactions allows.

Further advantages, features, properties and aspects of the present invention will become apparent from the claims and the following description of preferred embodiments with reference to the drawing. It shows:

1 shows a not to scale view of a part of a proposed device according to a first embodiment;

Fig. 2 is a schematic view of part of a proposal

Device according to a second embodiment; and

Fig. 3 is a not to scale view of a portion of a proposed device according to a third embodiment.

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

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

The proposed device 1 is used to examine a sample liquid 3, in particular by means of the ELIS A method. The following description is therefore directed essentially to the application or implementation of the ELISA method, where necessary, also supplementary or alternative measures or process steps can be performed. However, the proposed device 1 or the proposed method can in principle also be used for other examinations or methods.

Fig. 1 shows the sample liquid 3 immediately after filling in a first, common Aufhahmekammer 4. Several first metering chambers 5 - in the illustrated embodiment four first metering chambers 5a to 5d - are to the first Aufhahmekammer 4 by corresponding channels or the like. - In DarStellungsbeispiel by channel 18 - 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 metering chambers 5, wherein air and / or excess sample liquid 3 can continue to flow into an optional first collecting chamber 6. Thus, the channel 18 connects the first receiving chamber 4 with the first collecting chamber 6. FIG. 1 shows the device 1 in a state immediately after the sample liquid 3 has been introduced into the first receiving chamber 4, ie before the sample liquid 3 flows into the first metering chambers 5.

The device 1 has a second common receiving chamber 7 for receiving a diluting liquid 8. To the second receiving chamber 7 are a plurality of second metering chambers 9 - in the illustrated embodiment, four second metering chambers 9a to 9d - connected, which are also arranged in the illustration in a row and at least substantially parallel to the first metering chambers 5. The diluting liquid 8 flows via channel 19 into the second metering chambers 9. Excess diluting liquid 8 can, if required, flow into an optionally provided second collecting chamber 10. The channel 19 preferably connects the second Aufhahmekammer 7 with the second collection chamber 10. FIG. 1 shows the device 1 in the state immediately after the filling of the dilution liquid 8 in the second Aufhahmekammer 7, ie before the dilution liquid 8, the second metering chambers 9 and associated channels or the channel 19 and possibly the collection chamber 10 fills. The metering chambers 5 and 9 are preferably formed, for example by means not shown guide elements for the liquids 3 and 8, that the metering chambers 5, 9 and possibly the channels are completely filled without gas or air inclusion. Displaced air can escape via the preferably open collector chambers 6, 10 and / or via vent openings, not shown, which are associated in particular with the channels 18 and 19 and / or the metering chambers 5, 9.

The first and second metering chambers 5 and 9 are reaction chambers 11 - corresponding to the number of first and second metering chambers 5a to 5d and 9a to 9d in the illustrated embodiment, that is, four reaction chambers 11a to 11a

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

The first and second metering chambers 5, 9 are preferably assigned in pairs to each other and each one reaction chamber 11, each pair is fluidly connected to the associated reaction chamber 11 by corresponding, in particular radially extending, preferably channel-like compounds 12, for example, the first metering chamber 5b and second metering chamber 9b with the associated reaction chamber I Ib. The letters a to d thus indicate the assignment of the individual chambers 5, 9, 11 and 16 in the representation example. Accordingly, the liquid transfer takes place, in particular for dilution, mixing and / or reaction.

In the illustrated example, the first metering chambers 5 and 9 with the sample liquid 3 and the dilution liquid 8 preferably automatically fill themselves due to pressure and / or capillary forces, in particular when filling the liquid 3 or 8 in associated Aufhahmekammern 4 and 7 by means of a non-illustrated Pipette o. The like. And due to the pressure exerted on the liquid 3 and 8, respectively. However, other forces, possibly even centrifugal forces, depending on the arrangement and design, alternatively or additionally be used. The volumes of sample liquid 3 present in the first metering chambers 5 and the volumes of dilution liquid 8 present in the second metering chambers 9 can then be transferred into the respective associated reaction chamber 11 by appropriate centrifugal forces (caused by corresponding rotation of the device 1 about the axis of rotation 2). Thus, in the illustrated example, radial transfer takes place, the sample liquid 3 and dilution liquid 8 mixing in each case. However, in addition or as an alternative, other forces, for example pressure forces, capillary forces or the like, can act to convert the said volumes into the reaction chambers 11.

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

In particular, both the volumes of the first metering chambers 5 and the volumes of the second metering chambers 9 vary. For example, a first metering chamber 5d with a small volume is assigned a second metering chamber 9d with a large volume and vice versa. In the illustrated example, this is achieved by the volume of the first metering chambers 5 increasing or decreasing in a circumferential direction and the volumes of the second metering chambers 9 decreasing or increasing inversely in this circumferential direction. This allows a dilution series with a large dilution range - ie in particular from a low dilution ratio to a large 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 preferably, the sums of the volumes of the pairwise associated first and second metering chambers 5a and 9a, 5b and 9b, 5c and 9c and 5d and 9d are at least substantially equal. In this way, in addition to a particularly compact construction, it can be achieved that the individual volumes of differently diluted sample liquid 3 are of equal size and the reaction chambers 11 and any other downstream chambers or the like can be made uniformly large. In the previous and in the following description is always turned off on the respective volume of the metering chambers 5 and 9 respectively. To obtain accurate, defined dilution ratios, well-defined volumes are required. In order that, when transferring the sample liquid 3 and the dilution liquid 8 from the first and second metering chambers 5 and 9 into the associated reaction chambers 11, only defined volumes of the liquids 3, 8 are present and transferred and mixed, valve devices, obstacles or liquid stops, not shown, For example, to the connections 12 to the channels 18, 19 associated vents and / or the like. Provided.

In the illustrated embodiment, first separation points T _{a to} T _le for the liquid 3 in the first channel 18 is formed, in particular between the first Aufnah- measuring chamber 4 and the first metering chamber 5 a, between the individual dosing chambers 5 and between the last dosing chamber 5d, and the first plenum 6. Accordingly, second separation points T _2a to T _{2e are formed} for the liquid 8 in the second channel 19, in particular between the second receiving chamber 7 and the following second metering chamber 9a, see between the second metering chambers 9 and between the last metering chamber 9d and the second Receiving chamber 10. However, the first and second separation points T can alternatively or additionally be formed at the transition to individual chambers and / or other suitable locations.

Further, in the illustrated embodiment, preferably channel stops KSi and KS _{2 are formed} in the channel 18 and 19 between the last separation point Ti _e and T _2e and the respective collection chamber 6 or 10 or at the transition to the respective collection chamber 6 and 10, respectively Such flow resistance for the respective liquid 3 and 8 to form that when filling initially the first and second metering chambers 5 and 9 are completely filled with the respective liquid 3 and 8, before this in the associated collection chamber 6 or 10 can continue to flow.

In the illustrated embodiment, first liquid stops Si ₃ to S _d and second liquid stops S _2a to S _{2d are} preferably in the radially extending connections 12 between the respective first metering chambers 5 and the second metering chambers 9 and the second metering chambers 9 and the reaction chambers 11. However, these liquid stops S can alternatively or additionally also be formed at the transitions to the respective chambers.

The first liquid stops Si prevent the sample liquid 3 from undesirably filling the second metering chambers 9 when the first metering chambers 5 are filled. Conversely, the first liquid stops S] also prevent the diluting liquid 8 from undesirably filling the first metering chambers 5 or displacing the sample liquid 3 from the first metering chambers 5 when filling the second metering chambers 9. However, for this purpose, additional, not shown, liquid stops, for example, be provided at the transition of the compounds 12 in the respective second metering chambers 9.

The second liquid stops S ₂ prevent the dilution liquid 8 from undesirably flowing into the reaction chambers 11 during the filling of the second metering chambers 9, whereby a defined metering would no longer be possible.

The channel stops KS and the liquid stops S are formed in such a way, or tuned to the liquids 3 and 8 or to the particular during the filling by means not shown pipettes o. The like. Occurring pressures that the first and second liquid stops S ₁ and S _second during the filling of the first and second metering chambers 5, 9 with the liquids 3, 8 can not be overcome, but only in the later desired transfer of the individual volumes of liquid 3 and 8 from the metering chambers 5, 9 in the reaction chambers 11, in particular only with corresponding rotation of the device 1 or only with corresponding centrifugal forces. The liquid stops S are designed such that the second liquid stops S ₂ can be opened or overcome before the first liquid stops S]. This can also be achieved with the same or similar configuration and characteristic of the first and second liquid stops S, that greater centrifugal forces than in the first liquid stops Si occur at the second liquid stops S ₂ , the S radially outward relative to the first liquid Si stops or act. The separation points T and liquid stops S lead to defined volumes of liquid 3 and 8, which are mixed together. When transferring the liquid volumes from the first and second metering chambers 5, 9 into the reaction chamber 11, the liquid 3 or 8 breaks off at the separation points T and then flows via the respective, in particular radial, connection 12 into the associated reaction chamber 11 For example, the volume of liquid assigned to the second metering chamber 9b is determined or defined by the two second separation points T _2b and T _2c and the two liquid stops S _1b and _2b . The to be transferred and metered volume of sample liquid 3 is, for example, for the first metering chamber 5b through the two separation points T ₁ and T _b] _c and stop by the liquid Si _b limited. This applies correspondingly to the other liquid volumes of the further metering chambers 5, 9.

Preferably, the separation points T by corresponding, not shown ventilation o. The like. Formed. The liquid stops S and / or the channel stops KS are preferably formed by a corresponding constriction, sudden cross-sectional widening and / or modification of the wetting behavior, so that the respective liquid 3, 8, 14 can not or can not readily overcome the respective stop S, KS. Rather, in particular, a predetermined, for the individual stops S, KS, if necessary, different centrifugal force, compressive force o. The like. Required to overcome the respective stop S, KS can.

With regard to the necessary and / or possible structural 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 hereby introduces supplementary or alternatively as a disclosure becomes.

The above-described "parallel dilution" allows the generation of a dilution series in a single step, so that at best only slight dilution errors occur. In particular, this can be In addition, the problem of summation of single defects occurring in sequential dilution can be avoided.

In each reaction chamber 11, a desired reaction and in particular a plurality of desired reactions can then take place or be carried out, as will be discussed in more detail later. To carry out the ELISA method, the reaction chambers 11 are preferably first prepared before the diluted sample liquid 3 is supplied. This preparation takes place in particular before the sample liquid 3 is introduced into the first receiving chamber 4 and the diluting liquid 8 into 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 a sequential receptacle of different liquids 14, such as a reaction liquid, a washing liquid, a blocking or fixing liquid, a substrate liquid or the like. on. The reaction chambers 11 are connected to the third receiving chamber 13, so that in particular by pressure, capillary and / or centrifugal forces a filled into the receiving chamber 13 liquid 14 via corresponding channels or the like. - In the illustrated embodiment via a channel 20, preferably extends in the circumferential direction and / or parallel to the channels 18 and 19 - in the

Reaction chambers 11 can flow. Excessive and / or extruded

Liquid 14 is preferably collected in an optionally provided, third collection chamber 15, wherein an optimal channel stop KS ₃ can ensure that the liquid 14 completely fills the reaction chambers 11 before it flows into the third collection chamber 15.

In particular, the device 1 is embodied such that the third receiving chamber 13 is first completely emptied or emptied before another liquid 14 is supplied to the third receiving chamber 13, for example by pipetting. The emptying of the third Aufhahmekammer 13 can be achieved, for example, that after filling the third receiving chamber 13 with a liquid 14 by capillary forces automatically flows through the reaction chambers 11 and possibly the third collection chamber 15 until the third Aufhahmekammer 13 fully. is constantly emptied. Additionally or alternatively, this can be achieved by centrifugal forces, in particular in the case of a radial gradient (increase in the radial distance to the pivot point 2) of the channel 20 to the third collection chamber 15, and corresponding rotations of the device 1, and / or other forces.

In addition, the reaction chambers 11 can be emptied again, if necessary, before a new liquid 14 is introduced into the third receiving chamber 13 and this new liquid 14 flows into the reaction chambers 11. The prior emptying of the reaction chamber 11 is then preferably carried out by centrifugal forces, not shown valve means or the like. To allow a controlled emptying of the reaction chambers 11.

To prepare the reaction chambers 11 for the ELISA method, a liquid 14 containing a reagent, preferably an antibody, is first of all introduced into the third receiving chamber 13 and passed into the reaction chambers 11 in order to immobilize the reagent in the reaction chambers 11, in particular To bind the antibody in the appropriately prepared reaction chambers 11 and to coat the Reaktionskammem 11 with the antibody.

After a certain incubation or reaction time, the reaction chambers 11 are rinsed with a washing liquid, which is filled as the next liquid 14 in the third Aumahmekammer 13, to remove unbound reagent.

With a further liquid 14 follows, if necessary, a blockage of the still free, so in particular unoccupied by antibody binding sites to block a later Undefined binding of other reagents, or fixation of the immobilized reagent or the immobilized antibody in the reaction chambers 11th

After a possible rinsing with a washing liquid and if necessary

Empty the reaction chambers 11 are then prepared to the diluted sample liquid 3 - ie the sample liquid 3 and the Verdünnungsflüs- 8 from the associated first and second metering chambers 5 and 9 - record.

After transferring the sample liquid 3 together with the dilution liquid 8 into the reaction chambers 11, the actual detection reaction or a first reaction for examining the sample liquid 3 can take place. An analyte contained in the sample liquid 3 can bind in the illustrated embodiment, in particular to the immobilized reagent, in particular the immobilized antibody. After a preferably determined or defined reaction time unbound analyte is rinsed or washed from the reaction chambers 11, in particular by a single filling of a washing liquid 14 in the third Aufhahmekammer 13 to displace the existing liquids 3, 8 from the reaction chambers 11, and / or by centrifugal forces or other forces.

Subsequently, another liquid 14, which in particular contains an enzyme bound to a detection antibody, is fed to the reaction chambers 11, in that this liquid 14 is again supplied to the third receiving chamber 13. The detection antibody is designed such that it binds together with the enzyme to the complexes formed from the immobilized antibodies and the analyte in the reaction chambers 11.

Unbound detection antibodies and enzymes are subsequently rinsed out of the reaction chambers 11 in a washing step by preferably supplying a further washing liquid 14 once.

Finally, a substrate solution as further liquid 14 is preferably again supplied to the reaction chambers 11 via the third receiving chamber 13. The substrate is converted or modified by the enzymes in the reaction chambers 11 in an enzymatic detection reaction, so that a later detectable detection substrate, in particular a fluorescent or other dye or the like, is formed. The stopping of the detection reactions in the reaction chambers 11 and the further investigation will be discussed in more detail later. The supply of the various liquids 14, which preferably takes place exclusively via the common third receiving chamber 13 by sequential supply of the liquids 14, allows a very quick and easy preparation of the reaction chambers 11 and / or conducting the reactions in the reaction chambers 11, wherein the Pipettieraufwand , the required washing steps and / or the required amounts of liquid compared with the prior art - in particular over the conventional ELIS A method in an open pipetting plate - are significantly reduced.

So far, the running in the reaction chambers 11, already mentioned, in particular enzymatic or catalytic detection reactions by addition of an acid, a base or other stop solution or the like were stopped, for example by inactivation of the enzymes or catalytic reaction. This is possible in principle in the proposed device 1.

However, the detection reactions are particularly preferably stopped by separating the liquid with the substrate and the detection substrate from the (immobilized) enzymes, reaction catalysts or other reaction partners and / or additionally provided examination chambers 16, by the liquid in the reaction chambers 11 containing the substrate and the detection substrate to stop the detection reactions in each case in an associated examination chamber 16 is transferred. This transfer preferably takes place simultaneously for several or all reaction chambers 11, so that the detection reactions are stopped at the same time. In particular, the said transfer or stopping by centrifugal forces by the device 1 is rotated accordingly. However, the transfer is additionally or alternatively also possible by other forces, for example pressure or capillary forces, by means of corresponding valves or the like.

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 enables a very simple and highly simultaneous stopping of the detection reactions. NEN, so that over the prior art, a much more defined process flow and thus a much more accurate determination of the analyte are made possible.

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

Additionally or alternatively, the reaction chambers 11 may be associated with an optionally provided, indicated in Fig. 1 by dashed lines collecting channel 17, which is connected for example via the examination chambers 16 and corresponding, preferably radial connections 12 to the reaction chambers 11 in order to empty the reaction chambers 11 liquid from the reaction chambers 11, in particular when the reaction chambers 11 are emptied by centrifugal forces by corresponding rotation of the device 1. These liquids can then be discharged through the examination chambers 16 or through not shown, direct connections or the like. Into the collecting channel 17. Such an emptying of the reaction chambers 11 can be carried out, for example, to remove liquids 3, 8 and / or 14 before feeding a new liquid 14 into the reaction chambers 11.

In the illustrated example, third liquid stops S _3a to S _3C i are preferably formed in the (radial) connections 12 between the reaction chambers 11 and the checking chambers 16. The third liquid stops S ₃ can in particular, together with the second liquid stops S _2, prevent an undesired escape of the liquid 14 into other regions, so that the liquids 14 are desirably for example only in the third collection chamber 15 or, if necessary, when the third liquid stop S is overcome ₃ can be emptied or derived via the examination chambers 16 and optional fourth liquid stops S ₄ into the collecting channel 17. In particular, the third liquid stops S ₃ ensure a defined holding of the liquid volumes of liquids 3 and 8 transferred or metered into the reaction chambers 11, thus preventing uncontrolled and undesirable outflow from the reaction chambers 11.

In addition, separating points or liquid stops not shown in the channel 20 or in other connections between the reaction chambers 11 and / or to the third receiving chamber 13 or third collecting chamber 15 may be provided, as required, to prevent unwanted transfer of diluted sample liquid 3 from a reaction chamber 11 in an adjacent reaction chamber 11 - for example, when mixing by accelerating and decelerating - to be able to prevent.

In addition or as an alternative, the channel 20 and, in particular, its sections extending between the individual reaction chambers 11 may also have a different course, deviating in the radial direction, from the course with an at least substantially constant distance or radius to the pivot point 2, in order to prevent unwanted transfer of dilute sample liquid 3 between individual reaction chambers 11 to prevent. The same applies to the other channels 18 and 19 and the respective channel sections between the metering chambers 5 and 9, respectively.

Preferably, fourth liquid stops S _4a to S _{4d are arranged} in the radial connections 12 between the examination chambers 16 and the optional collection channel 17, in order to prevent an undefined outflow or discharge of liquid from the examination chambers 16.

The third and fourth liquid stops S ₃ and S ₄ can in turn also be formed, as required, at the transitions from the reaction chambers 11 and 16 to the respective connections 12.

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, in particular about 10, dilutions or different dilution ratios are produced. Of course, several can parallel dilutions occur simultaneously on the device 1. Accordingly, the device 1 may, if necessary, also have a plurality of arrangements, as shown in FIG.

Subsequently, a second embodiment of the proposed device 1 and the proposed method will be explained in more detail with reference to FIG. 2, wherein the following explanations 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 illustration according to FIG. 2, the preferably provided curvature has been omitted in the preferably provided ring structure for placement on a round disk, such as a CD or the like, in order to allow a better clarity. Next, the illustration of FIG. 2 is 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 the case in the illustration according to FIG. 1.

In Fig. 2, for simplicity reasons, also no liquids 3, 8, 14 are shown. However, the relevant explanations in the first embodiment and also with regard to the other method sequence apply correspondingly to the second embodiment shown in FIG. Furthermore, in FIG. 2, for reasons of simplification, the optional collecting channel 17 has been omitted.

In FIG. 2, for reasons of simplification, no separation points T ₅ liquid stops S and channel stops KS are shown. However, the explanations and arrangements relating to this in the first embodiment apply correspondingly or additionally to the second embodiment.

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

The further parallel dilution takes place substantially in accordance with 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 the additional parallel dilution, therefore, additional first metering chambers 5 'and additional second metering chambers 9' are provided. and additional reaction chambers 11 'are provided. The additional metering 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.

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

The transfer of the individual liquid volumes into the associated additional reaction chambers 11 'is again preferably effected 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, another additional or additional dilution liquid can be separately supplied to the additional second metering chambers 9 'via an additional receiving chamber (not shown) for further dilution.

If only a partial further dilution takes place, as shown in FIG. 2, it is preferable, but not necessarily, for those reaction chambers 11, the contents of which are not further diluted, each associated with additional reaction chambers IT, which are arranged in particular to a corresponding extent as the further dilution serving additional reaction chambers 11 'in order for all dilution stages a simultaneous examination, in particular binding of the analyte the immobilized reagent to ensure or facilitate.

Optionally, an additional first collection chamber 6 'may be provided, which is connected to receive additional sample liquid 3 to the additional first metering chambers 5'. Optionally, an additional second collection chamber 10 'may be provided, which is arranged to receive excess dilution liquid 8 at the additional second metering chambers 9'.

Hereinafter, a third embodiment of the proposed device 1 and the proposed method will be explained in more detail with reference to FIG. 3, wherein the following explanations are limited only to significant differences from the first and second embodiments. The previous explanations thus apply in addition or in a corresponding manner.

In the third embodiment, the first metering chambers 5 are connected in parallel to a first, in particular common channel 18, which leads from the first receiving chamber 4 to the first collecting chamber 6. This has the advantage that a faster filling of the first metering chambers 5 with sample liquid 3 is possible, since these are parallel, that is, can be charged at the same time. In particular, a filling by pressure, for example by attaching a pipette, not shown, or the like., To the first, open receiving chamber 4, wherein thereby taking place (partial) filling of the first collection is melamine chamber 6 with appropriate dimensioning uncritical.

The first channel 18 is emptied after filling the first metering chamber 5 - in particular by capillary and / or centrifugal forces - before transferring the sample liquid 3 from the first metering chambers 5 into the associated reaction chambers 11 into the first collecting chamber 6. This leads to a particularly accurate metering, since such a defined "tearing off" of the sample liquid 3 at the transitions (separation points Ti) from the channel 18 to the individual first metering chambers 5 or corresponding connections is achieved. This allows particularly accurate dosages, which then lead to correspondingly precise dilution series in the subsequent mixing with dilution liquid 8 and in particular in the ELISA method to very accurate quantitative results.

The second metering chambers 9 are preferably connected in a corresponding manner parallel to a second, in particular common, channel 19, which connects the second receiving chamber 7 to the second collecting chamber 10. Accordingly, the second metering chambers 9 can be filled faster with the diluting liquid 8. Preferably, the filling with the diluting liquid 8 also follows by pressure, in particular by applying a pipette, not shown, or the like.

Furthermore, the second channel 19 is preferably completely emptied into the second collection chamber 10 after filling the second metering chambers 9, in particular by capillary and / or centrifugal forces, before the dilution liquid 8 is transferred from the second metering chambers 9 into the associated reaction chambers 11 becomes. This in turn results in a very accurate dosage, since the dilution liquid 8 defined at the transitions (separation points T ₂ ) from the channel 19 to the metering chambers 9 and corresponding connections tears off, as already explained above for the sample liquid 3 and the first metering chambers 5. Accordingly, this allows particularly accurate dilution series and in particular very accurate quantitative investigations according to the ELISA method or in any other way. The first and second channels 18, 19 are preferably emptied simultaneously.

The separation points T are formed in particular by corresponding constrictions and / or kinks to ensure the desired defined tearing of the liquid.

The parallel connection provided in the third embodiment of the first metering chambers 5 to the first channel 18 and / or the second metering chambers 9 to the second channel 19 allows, as already explained, a particularly fast and parallel filling of the chambers 5 and 9 and is required Also, independently of other aspects and features of the present Ausruhrungsformen feasible.

The channels 18 and 19 preferably have in turn channel stops KS ₁ and KS ₂ to the respective collection chamber 6 and 10 down to ensure that first the respective metering chambers 5 and 9 are completely filled before the corresponding liquid 3 and 8 in the associated collection chamber 6 and 10 can continue to flow. In particular, the channel stops KS are designed such that they are supplied by the respective liquid 3 or 8 of the pressure for feeding - for example, by a pipette, not shown, with which the respective liquid is supplied to the associated Aumahmekammer 4 and 7 - (first) can be overcome after complete filling of the associated metering chambers 5 and 9 respectively. Thus, complete filling of the metering chambers 5 and 9 with the respective liquid 3 or 8 can be ensured.

In order to enable or assist complete emptying, the channels 18 and 19 are preferably substantially rectilinear or with only minor dislocations or kinks and / or preferably without V or U-shaped arcs. In order to facilitate or assist in complete emptying, the channels 18 and 19 alternatively or additionally preferably have a radial gradient, in particular between the respective start and end or the respective Aumahmekammer 4 and 7 and 6 and 10 collecting chamber, so that the centrifugal forces increasing with increasing radius lead, with appropriate rotation of the device 1, to the desired emptying of the channels 18, 19.

In the third embodiment, the associated first metering chambers 5 and second metering chambers 9 are not connected in series as in the first or second embodiment (the order being freely selectable) or connected in series to the associated reaction chambers 11, but preferably in parallel or quasi connected in parallel to the associated reaction chambers 11. Particularly preferred is the "quasi-parallel" connection, which is explained in more detail below with reference to FIG. 3. The second metering chambers 9 are connected via connections 12, which preferably extend at least substantially radially, to the associated reaction chambers 11. The second liquid stops S ₂ prevent an uncontrolled flow of the diluting liquid 8 from the second metering chambers 9 via the connections 12 into the reaction chambers 11.

The first metering chambers 5 are now in turn - preferably via first liquid stops Si - connected to the associated compounds 12, in particular after each of the second liquid stops S ₂ . The first liquid stops Si are each formed, for example, by a corresponding constriction or sudden cross-sectional widening, so that the sample liquid 3 from the first metering chambers 5 - preferably even upon reaching an angular velocity or centrifugal force, which leads to a transfer of the diluting liquid 8 from the second metering chambers 9 leads into the associated reaction chambers 11 - not or not readily transferred to the associated reaction chambers 11 via the connections 12. Rather, preferably a downstream wetting - in particular the liquid stops Si - by the dilution liquid 8 is required. Only then can the sample liquid 3 overcome the liquid stops Si or other connections to the connections 12 and then flow together with the dilution liquid 8 into the associated reaction chambers 11. The lateral or parallel introduction of the sample liquid 3 into the dilution liquid streams already leads to a first mixing or to a better mixing, so that in particular in the reaction chambers 11 a very good mixing can be achieved.

The preferred special training (rejuvenation) of the liquid stops S can also be omitted if necessary. Alternatively, not shown valve devices may be used instead.

Furthermore, it is also possible for the transfer of the dilution liquid 8 from the second metering chambers 9 on the one hand and the sample liquid 3 from the first metering chambers 5 - in particular when a certain angular velocity or centrifuge force is reached and exceeded - to occur virtually simultaneously. In this case also a (first) Mixing of the liquids 3, 8 achieved by the addition of the sample liquid 3 in the dilution liquid streams in the compounds 12.

If required, the feed can also be reversed, ie the dilution liquid 8 can be introduced into sample liquid streams in the connections 12. The above statements apply accordingly.

In the third embodiment, it is not decisive whether the first liquid stops S ₁ or the second liquid stops S _{2 are} first overcome by the respective liquid 3 or 8, since in both cases thorough mixing of the two liquids 3 and 8 - At least in the reaction chambers 11 - can be reached. Accordingly, the third embodiment is a very robust system.

Another aspect of the third embodiment is that, for example, the channels 18, 19, but also other cavities, connections 12 or the like., Not always on a flat side of the carrier - especially not on the flat side, in which the chambers 4 to 7, 9 to 11, 13, 15 and 16 - must be formed, in which the cavities, channels or the like. Are formed. Rather, in the illustration according to FIG. 3, the sections indicated by dashed lines are preferably formed on the underside, while the solid cavities, channels and the like are preferably formed on the upper side or from the upper side. The upper and lower side cavities, channels and the like are then connected to one another by means of corresponding apertures, bores or the like. This allows much greater freedom in the design of the device 1, in particular with regard to the arrangement, configuration and connection of the chambers. The cavities, channels and the like, which are preferably formed by the flat sides (upper and lower sides), are then covered on each flat side, preferably by a cover (not shown), for example a foil or disk, so that an at least quasi-closed system is formed , Only the required openings, for example for filling the chambers 4, 7, 13 and for venting or the like. Then then, possibly even closable, openings to the environment.

In the third embodiment, the reaction chambers 11 are not shown to scale. It should also be noted that the volumes of individual NEN chambers can also vary greatly depending on the depth of the chambers. Furthermore, examination chambers 16 according to the first or second embodiment can of course also be connected to the reaction chambers 11 if required.

Particularly preferably, the device 1 according to a feasible also independent of the present embodiment aspect of the present invention of several, preferably segmental modules M constructed, which can be arranged for example by means of an adapter or holder, not shown, in a disc-shaped configuration. This modular design allows the combination of different examinations as needed. In Fig. 3, only a single module M is shown schematically.

The individual features and aspects of the first, second and third embodiments can also be combined as desired. Furthermore, individual aspects can also be used independently of the present embodiments in other embodiments or applications.

The mixing of the sample liquid 3 with the dilution liquid 8 - especially in the reaction chambers 11 - can be required or achieved by braking and accelerating the rotation of the device 1.

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, that is to say in particular of the described chambers, channels, connections and the like, is preferably 20 to 1000 μm, in particular approximately 200 μm, in the illustrated embodiment.

All microstructures are preferably covered by a suitable, not shown or transparent cover. Only the receiving chambers 4, 7 and 13, possibly the collecting chambers 6, 10, 15, if necessary, the collecting channel 17 and / or other not shown vent openings or the like are formed open to the outside. Thus, the evaporation losses can be minimized and accordingly work with high accuracy with low volumes of liquid.

The volumes of liquids to be used are about 10 to 2,000 μl per liquid, 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 .mu.l, in particular about 10 ul. The same applies to the volumes of the reaction chambers 11 and the examination chambers 16. In particular, the said sum and the respective volumes of the reaction chambers 11 and the examination chambers 16 are the same.

In addition or as an alternative to the dilution of the sample liquid 3 by the dilution liquid 8, it is also possible to mix any liquids 3 and 8, ie, for example, two liquids 3 and 8 which react with one another. In particular, it may then be a reaction liquid 8 or the like instead of the dilution liquid 8. Accordingly, the terms "sample liquid" and "dilution liquid" are preferably also very generally understood as different liquids.

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 precise examination in the form of a precise quantitative determination of an analyte in the sample liquid is made possible.

Claims

claims:

1. Device (1) for examining a sample liquid (3), in particular by means of the ELISA method, with a first, common Aumahmekammer (4) for receiving the sample liquid (3), with a plurality of first metering chambers (5) for receiving Sample liquid (3), in particular by pressure and / or capillary forces, to the first receiving chamber (4) are connected to a second, common receiving chamber (7) for receiving a dilution liquid (8), with a plurality of second metering chambers (9) , which are connected to the second Aufhahmekammer (7) for the exclusive inclusion of dilution liquid (8), in particular by pressure, capillary and / or centrifugal forces, with a plurality of reaction chambers (11), wherein the first and / or second metering chambers (5 , 9) vary in their volumes, the first and second metering chambers (5, 9) being associated in pairs with each other and each pair being connected to an associated reaction chamber (11) i st, so that by pressure and / or centrifugal forces contained in the first and second metering chambers (5, 9) volumes of sample liquid (3) and dilution liquid (8) in pairs in the associated reaction chambers (11) are überfuhrbar and miscible, whereby the Sample liquid (3) can be diluted with different dilution ratios.

2. Apparatus according to claim 1, characterized in that the volumes of the first metering chambers (5), in particular starting from the first receiving chamber (4), increase or decrease and the volumes of the second metering chambers (9) opposite to the volumes of decrease or increase assigned first metering chambers (5).

3. Apparatus according to claim 1 or 2, characterized in that the sums of each pairwise associated volumes of the first and second metering chambers (5, 9) are the same.

4. Device according to one of the preceding claims, characterized in that the diluted sample liquid (3) from at least one of the reaction chambers (11) is further dilutable.

5. Device according to one of the preceding claims, characterized in that the first metering chambers (5) connected in parallel, in particular via a first channel (18) to the first, common Aufhahmekammer (4) for receiving the sample liquid (3) are, in particular wherein the first channel (18) before the transfer of the sample liquid (3) from the first metering chambers (5) in the associated reaction chambers (11) can be emptied.

6. Device according to one of the preceding claims, characterized in that the second metering chambers (9) in parallel, in particular via a second channel (19) to the second, common Aumahmekammer (7) for receiving the dilution liquid (8) are connected, in particular wherein the second channel (19) prior to transfer of the dilution liquid (8) from the second metering chambers (9) in the associated reaction chambers

(11) is emptied.

7. Device according to one of the preceding claims, characterized in that each second metering chamber (9) via a connection (12) to an associated reaction chamber (11) is connected and each associated first metering chamber (5) in parallel to the associated reaction chamber ( 11) or - in particular via a liquid stop (S ₁ ) - to this compound

(12) is connected, preferably wherein the dilution liquid (8) in the transfer from the second metering chamber (9) into the reaction chamber (11) the outlet or liquid stop (S]) of the associated first metering chamber (5) wetted downstream of the transfer to assist the sample liquid (3) from the first metering chamber (5) into the reaction chamber (11) and in particular to overcome the liquid stop (S ₁ ) and / or to mix the sample liquid (3) with the diluent liquid (8), in particular in the section of the connection (12) from the connection of the first metering chamber (5) to the reaction chamber (11).

8. Device according to one of the preceding claims, characterized in that at least one of the reaction chambers (11) or parallel to a plurality of reaction chambers (11) each additional first metering chambers (5 ') for receiving diluted sample liquid (3), in particular by Pressure, centrifugal and / or capillary forces are connected, and that additional second metering chambers (9 ') for receiving dilution liquid (8), in particular by pressure, centrifugal and / or capillary forces, to the second receiving chamber (7), a collecting chamber (10) for diluting liquid (8) associated with the second metering chambers (9) or an additional feed of diluting liquid (8) are connected, the additional first and / or second metering chambers (5 ', 9') in their volumes vary, wherein the additional first and second metering chambers (5 ¹ , 9 ') are associated with each other in pairs and wherein each pair with an associated z is connected to the additional reaction chamber (H ') so that the volumes of already diluted sample liquid (3) and dilution liquid (8) contained in the additional first and second metering chambers (5', 9 ') are combined by pressure and / or centrifugal forces. in pairs in the associated additional reaction chambers (H ') are transferable and miscible, whereby the already diluted once sample liquid (3) with different dilution ratios can be further diluted.

9. Apparatus according to claim 8, characterized in that the volumes of the additional first metering chambers (5 ¹ ) increase or decrease and the volumes of the additional second metering chambers (9 ') opposite to the volumes of the associated additional first metering chambers (5') from - or increase.

10. The device according to claim 9, characterized in that the sums of each pairwise associated volumes of the additional first and second metering chambers (5 ', 9') are the same. 11. Device according to one of the preceding claims, characterized in that the device (1) has a third Aufhahmekammer (13) for in particular sequential recording one or more liquids (14), such as a liquid with a reagent, in particular an antibody, a washing liquid, such as a blocking liquid, in particular for blocking free binding sites in the reaction chambers (11,

11 '), such as a liquid having an enzyme bound to a detection antibody in particular, and / or a liquid having a substrate.

12. Device (1) for examining a sample liquid (3), in particular by means of the ELISA method, preferably according to one of the preceding claims, with a first Aufhahmekammer (4) for receiving the sample liquid

(3), with a third receiving chamber (13) for the sequential recording of different liquids (14), and with a plurality of reaction chambers (11, 11 ') to which the sample liquid (3) and the various liquids (14) are successively fed, the Device (1) is designed such that the third Aufhahme- chamber (13) is emptied each before a new intake of a liquid (14).

13. The apparatus according to claim 12, characterized in that the device (1) is designed such that a liquid with a reagent, in particular an antibody, a washing liquid, such as a blocking liquid, in particular for blocking free binding sites in the reaction chambers (11 , 11 '), such as a liquid with an enzyme, which is in particular bound to a detection antibody, and / or a liquid with a substrate of the third receiving chamber (14) is receivable or are.

14. Device according to one of claims 11 to 13, characterized in that several or all reaction chambers (11) for sequential Aufhah- me the liquid (s) (14) by pressure, capillary and / or centrifugal forces to the third receiving chamber (13) are connected.

15. Device according to one of the preceding claims, characterized in that the device (1) at least one of the reaction chambers (11, 11 ') and / or downstream examination chambers (16) associated collecting chamber (15) and / or a collecting channel (17 ) for receiving the liquids (3, 8, 14).

16. Device according to one of the preceding claims, characterized in that the device (1) the reaction chambers (11) and / or additional reaction chambers (H ') associated with examination chambers (16), so that in the reaction chambers (11, 11') expiring detection reactions in particular can be stopped simultaneously by in the reaction chambers (11, 11 ') located liquids (14), in particular by pressure, capillary and / or centrifugal forces, respectively in the associated examination chambers (16), in particular simultaneously be transferred , in particular wherein in the reaction chambers (11, 11 ') immobilized reagents, such as enzymes, are arranged.

17. Device according to one of the preceding claims, characterized in that the device (1) as a microfluidic system, in particular in the form of a round disc, such as a CD, formed and / or preferably segmental modules (M) is constructed.

18. A method for examining a sample liquid (3), in particular by means of the ELISA method, wherein the sample liquid (3) is fed to a first, common receiving chamber (4) and forwarded from this into a plurality of first metering chambers (5), wherein a dilution liquid ( 8) is fed to a second, common receiving chamber (7) and forwarded from it into a plurality of second metering chambers (9), wherein the first and / or second metering chambers (5, 9) vary in their volumes, the first and second metering chambers (7) 5, 9) in pairs are assigned by pressure and / or centrifugal forces in the first and second metering chambers (5, 9) contained volumes of sample liquid (3) and dilution liquid (8) in pairs in associated reaction chambers (11) and mixed, whereby the sample liquid ( 3) diluted with different dilution ratios and then examined.

19. The method according to claim 18, characterized in that a first channel (18) to which the first metering chambers (5) for supplying the Probenflüs- sigkeit (3) of the first receiving chamber (4) are connected, is first emptied before the sample liquid (3) from the first metering chambers (5) in the associated reaction chambers (11) is transferred, and / or that a second channel (19) to which the second metering chambers (9) for supplying the diluting liquid (8) of the second receiving chamber (4) are connected, is first emptied before the sample liquid (3) from the second metering chambers (9) is transferred into the associated reaction chambers (11).

20. The method according to claim 18 or 19, characterized in that during or during the transfer of the sample liquid (3) from the first metering chamber (5) in the associated reaction chamber (11) via a connection (12), this compound from the dilution liquid (8 ) is flowed through in the transfer of the associated second metering chamber (9) in the reaction chamber (11), preferably wherein the dilution liquid (8) during the transfer of the second metering chamber (9) into the reaction chamber (11) the connection of an associated the first metering chamber (5) wetted downstream, in order to assist the transfer of the sample liquid (3) from the first metering chamber (5) into the reaction chamber (11) and in particular to overcome a capillary stop (20).

21. The method according to any one of claims 18 to 20, characterized in that during the transfer of the sample liquid (3) and the dilution liquid (8) from the metering chambers (5, 9) in the associated reaction chamber (11) via respective connections (12). the sample liquid (3) laterally in the through the compounds (12) flowing dilution liquid (8) or vice versa is introduced, in particular in order to mix the liquids (3, 8) already in the compounds (12).

22. The method according to any one of claims 18 to 21, characterized in that the sample liquid (3) and the dilution liquid (8) in parallel or simultaneously in the respective reaction chamber (11) are transferred in particular for better mixing.

23. The method according to any one of claims 18 to 23, characterized in that the diluted sample liquid (3) from at least one of the reaction chambers (11) is further diluted.

24. The method according to claim 23, characterized in that for further dilution, the already diluted once sample liquid (3) from a reaction chamber (11) to additional first metering chambers (5 ¹ ) is passed and that the dilution liquid (8) to additional second Dosing chambers (9 ') is passed, wherein the additional first and / or second metering chambers (5', 9 ') vary in their volumes and the additional first and second metering chambers (5', 9 ') are associated in pairs, so that by Pressure and / or centrifugal forces in the additional first and second metering chambers (5 ', 9') contained volumes of already diluted sample liquid (3) and dilution liquid (8) in pairs in associated additional reaction chambers (H ¹ ) are transferred and mixed, whereby the already diluted once sample liquid (3) with different dilution ratios further diluted and then examined becomes.

25. A method for examining a sample liquid (3), in particular by means of the ELISA method, preferably according to one of claims 18 to 24, wherein in reaction chambers (11, H ¹ ) detection reactions for the detection of an analyte in the sample liquid (3) proceed, wherein the succession of pressure, centrifugal and / or capillary forces to the reaction chambers (11, 11 ') via a common third receiving chamber (13), wherein the third receiving chamber (13) is automatically emptied by capillary forces and / or by centrifugal forces before each receiving a new liquid (14).

26. The method according to any one of claims 18 to 25, characterized in that the reaction chambers (11, IT) successively different liquids (14), such as a liquid with a reagent, in particular an antibody, such as a washing liquid, such as a blocking liquid, in particular for Blocking of free binding sites in the reaction chambers (11, IT), such as a liquid with an enzyme, in particular bound to a detection antibody, and / or as a liquid with a substrate, preferably via a common third receiving chamber (13), in particular by pressure -, capillary and / or centrifugal forces are supplied.

27. The method according to claim 25 or 26, characterized in that the third receiving chamber (13) is emptied each before receiving a new liquid (14), in particular automatically by capillary forces and / or by centrifugal forces.

28. The method according to any one of claims 18 to 27, characterized in that the reaction chambers (11, 11 ') before supplying the sample liquid (3) with a preferably immobilized reagent, in particular an antibody, provided, in particular coated.

29. The method according to claim 28, characterized in that after immobilization of the reagent still free binding sites in the reaction chambers (11, 11 ') are blocked by means of a blocking liquid.

30. The method according to claim 28 or 29, characterized in that each after the coating and / or blocking, in particular only once, a washing liquid for rinsing through the reaction chambers (11, 11 ') is passed.

31. The method according to any one of claims 28 to 30, characterized in that then the Probenfiüssigkeit (3) with the respective dilution ratio, in particular by pressure, capillary and / or centrifugal forces, into the respective reaction chamber (11, 11 ') is passed.

32. The method according to claim 31, characterized in that after a certain reaction time and / or binding of an analyte in the sample liquid (3) to the reagent, the reaction chambers (11, 11 ') are rinsed, then to the compound of reagent and analyte-binding detection reagent, in particular a detection antibody with an enzyme, into the reaction chambers (11, 11 ') is passed and finally the unbound detection reagent or unbound enzyme is washed out again.

33. The method according to claim 32, characterized in that after washing out of the unbound detection reagent or unbound enzyme, a substrate in the reaction chambers (11, 11 ') is passed, of the detection reagent or enzyme, in particular enzymatically, in a detection reaction in an analyte is modified or converted.

34. The method according to any one of claims 18 to 33, characterized in that in the reaction chambers (11, 11 ') runs a particular enzymatic or other catalytic detection reaction for the detection of an analyte in the sample liquid (3) and thereby a detection substrate is produced.

35. The method according to any one of claims 32 to 34, characterized in that the detection reaction in several or all reaction chambers (11, 11 ') is stopped simultaneously by the in the respective reaction chambers (11, 11') located liquid (14) the substrate and the detection substrate from the reaction chambers (11, 11 ') in associated examination chambers (16), preferably by pressure, capillary and / or centrifugal forces, überfuhrt and thereby required for the detection reaction reactants are separated.

36. The method according to any one of claims 18 to 35, characterized in that the method in a microfluidic system, in particular in the form of a round disc, such as a CD ₅ and / or in a structure of preferably segmental modules (M) is performed, in particular in which to generate centrifugal forces for controlling the process sequence, the microfluidic system is rotated at specific times, for specific periods of time and / or at specific rotational speeds.