WO2006056001A1

WO2006056001A1 - Device for qualitatively and quantitatively analyzing analytes

Info

Publication number: WO2006056001A1
Application number: PCT/AT2005/000477
Authority: WO
Inventors: Thomas Schlederer
Original assignee: Thomas Schlederer
Priority date: 2004-11-25
Filing date: 2005-11-25
Publication date: 2006-06-01

Abstract

The invention relates to a device for analyzing at least one analyte in a sample, comprising a distributor (1) with at least one inlet opening (2-6) for introducing liquids, suspensions and/or gases into the distributor (1), and with at least one outlet opening (9) for removing liquids, suspensions and/or gases from the distributor (1). To this end, a tubular hollow body (10), which is connected to the distributor (1), is attached to at least one outlet opening (9). A magnetic device (11) is arranged on said hollow body while serving to temporarily immobilize magnetic analyte-binding particles, which are introduced into the hollow body (10) or located inside the hollow body (10), on an inner delimitation of the hollow body (10).

Description

Device for the qualitative and quantitative determination of analytes

The present invention relates to a device for the qualitative and quantitative determination of analytes.

The determination of clinical parameters in a simple, reliable and fast manner is the goal of "near patient testing" (NPT) methods and devices outside the clinical laboratory in the corresponding hospital wards, doctor's offices or at the patient's home Devices thus close the gap between the patients, the treating physicians and the large clinical laboratories.

Areas where such procedures may be used include the areas of blood clotting, emergency diagnostics, markers of heart disease (heart attack), urinalysis, clinical chemistry, immunology, and metabolic disorders.

While the requirements of the NPT, such as low sample volume, simple testing and short analysis times, are met for the areas of coagulation, blood gas analysis, myocardial infarction diagnosis and urinalysis, the known solutions are unsatisfactory in the field of immunology or emergency medicine.

The US 6,133,043 relates to a device for analyzing a sample comprising a cell having a defined volume and an electrode which is capable of applying an electrical voltage to the sample, a magnetic Auffangvor¬ direction with one or more magnets, a voltage source for generating a Voltage at the electrode sufficient to induce luminescence of an electrochemiluminescent substance in the sample, and a light detector.

The device comprises inter alia a detection device coupled to a measuring cell, a distributor and a pump. The distributor has a plurality of inlet openings and an outlet opening for distributing various solutions (including the sample mixture itself) into the tubular hollow bodies connected to the distributor, which are connected to the pump via the magnetic device and the detection device. Such an arrangement leads to a high mechanical load of any magnetic particles used. In US 2003/0127396 an apparatus for mixing and separating magnetic particles is disclosed. This device comprises, inter alia, a container which serves for receiving liquids and magnetic particles, and a magnetic device in the immediate vicinity of the container. In this container, one or more tubes can be inserted through an opening at the top, with the help of which washing liquids are transported in and out of the container.

In US 5,660,990 a general method for the qualitative or quantitative determination of an analyte in a sample is disclosed, which provides for the use of magnetic particles for the immobilization of this analyte.

WO 2004/011146 describes an apparatus for mixing, separating, collecting and washing magnetic particles, which includes, inter alia, a pipette whose upper end is arranged on a pressure device and a magnet.

WO 1994/11078 relates to a device for immobilizing and isolating cells or analytes bound to magnetic particles from a liquid medium. This device comprises inter alia magnetic means for generating a magnetic field and tubular hollow bodies for transporting the liquid medium.

EP 0 687 501 B1 discloses a method and a device in which magnetic particles in pipettes can be temporarily immo-stabilized during an examination with the aid of a magnet.

It is therefore an object of the present invention to provide a device or a method in order to meet the requirements of the clinic, especially in the field of intensive care and emergency laboratory, and to provide examination possibilities in order to obtain as many samples as possible be available in a small amount zu¬ reliable and easy to analyze in a few steps, with a full automation should be possible. As a result, the results of the NPT can be applied quickly and directly in the operating theater, in intensive care units, outpatient clinics, blood banks or directly in the doctor's office. Furthermore, it is also important for clinical practice that a large number of analytes can be determined with a single device.

Therefore, the present invention relates to a device for determining at least one analyte in a sample comprising a distributor having at least one inlet opening for introducing liquids, suspensions and / or gases into the distributor and having at least one outlet opening for removing liquids, suspensions and / or gases from the distributor in which a tubular hollow body connected to the distributor is attached to at least one outlet opening, to which a magnetic device for temporary immobilization of magnetic analyte-binding particles introduced into the interior of the hollow body through an inlet opening or inside the hollow body an inner boundary of the hollow body is arranged, wherein the distributor a displacer is arranged.

"Liquids and / or gases" refers to all those liquids that can be used in an analysis, which apart from the sample liquid are usually salt solutions, organic solutions, buffers, water, suspensions etc. or air, noble gases, inert Gases, etc., on the one hand serve to strengthen the binding analyte particles and clean the Vor¬ direction and on the other hand are able to displace any liquid from the device.

"Tubular hollow bodies" are tubular hollow bodies which are open at at least two ends and are also used, for example, in chromatography .These as well as all those components which come into contact with the fluids used should be inert to the solutions used the tubular hollow body, for example, consist of metal, but appropriate plastic is preferred.

"Analytically binding particles" are magnetic particles which are capable of specifically binding an analyte from a sample.The particles can have paramagnetic, superparamagnetic or ferromagnetic properties according to the invention, with paramagnetic and superparamagnetic particles preferably being used.

Magnetic particles can be immobilized by the application of a magnetic field on a surface and optionally concentrated, whereby the handling of such particles within the device is facilitated. Particles which are particularly suitable for the use according to the invention are described, for example, in "Biomagnetic Techniques in Molecular Biotechnology". logy ", Dynal, Technical Handbook 3 ^rd Edition © 1998.

Due to the apparatus construction of the device according to the invention, an automation is feasible throughout since all components (distributor, magnetic device and detector) can be controlled. A miniaturization of the device is possible in a simple manner, since the individual device parts can also be miniaturized. Both factors are particularly crucial for practical use, since small automated devices are particularly preferred by the user of the device according to the invention, since these are easily transportable, require small sample volumes and can be used in combination with other devices (eg blood gas machines). ,

According to the invention, an outlet opening can also serve to remove waste liquid from the device and to sufficiently flush the lines before using the device.

The inlet and outlet openings are preferably designed such that they are optionally closable, in order to prevent the escape of liquid or gas from the device during the changeover of the distributor when not using an opening. The closing could be carried out, for example, by attaching a valve.

In many respects, a device according to the invention has advantages over hitherto known devices for determining analytes to which magnetic particles are also used for binding the analytes to be examined. Thus, in the device "Pathfast" from Mitsubishi, which is based on the so-called "Magtration" (EP 0 687 501 B1) technology, the magnetic particles are temporarily immobilized in pipettes during the washing steps, the incubation of the particles with the sample, with a second label-conjugated analyte-binding partner and the subsequent detection reaction in containers. Above all, this device has the disadvantage that a correspondingly large sample volume and many pipetting steps are required, for which new containers with the corresponding liquids (eg washing solutions) must be made available in each case. In contrast, with the device according to the invention, small sample volumes can be carried out in one and the same hollow body. According to the invention, a displacer is arranged on the distributor. This can be connected to the distributor via its own positive displacement inlet opening.

A displacer serves to introduce a liquid or a gas into the device or to remove. In this case, the displacer is preferably attached to the distributor at an inlet opening (so-called displacement inlet opening) via a further tubular hollow body. The displacer should be able to move the substances to be transported in the tubular hollow bodies both forward and backward in order, if appropriate, to permit mixing of the magnetic particles in the tubular hollow bodies which are arranged at an opening. The arrangement of the displacer directly on the distributor via its own displacer input opening has a number of further advantages. Above all, it is possible during the forward and backward movement of the suspensions / solutions in Vorrich¬ device to reduce the risk of contamination of the solution container or Probenbe¬ container drastically, without introducing, for example, air into the device. Furthermore, by means of a der¬ like embodiment, it is possible to serially connect a plurality of measuring regions or measuring chambers and to further process or investigate the analytes bound to the magnetic particles outside the device, since the other flow movements can be left uninfluenced. Furthermore, by such an embodiment, the magnetic particles are not mechanically stressed significantly. As a result, the magnetic particles can be reused after any regeneration and washing steps, which, in particular, can significantly extend the product lifetime.

According to a preferred embodiment, the displacer is designed as a pump or syringe.

It has been found that, in particular, pumps and syringes are particularly well suited for bringing liquids into the device according to the invention and for moving them within them, in particular commercially available syringes and pumps, which are also used in chromatography can.

Preferably, the distributor is designed as a rotary or multi-way valve.

Rotary and multi-way valves are characterized by the many Due to the large number of openings, the distribution options are mainly due to their high degree of automation. Therefore, such valves are particularly well liquids and gases to distribute in the device.

Combinations of several distributors are also possible according to the invention, as a result of which mutual contamination of various liquids can be completely excluded. Furthermore, it is also possible to carry out individual work steps in parallel.

According to a further preferred embodiment, the distributor comprises a mixing chamber.

By providing a mixing chamber on or in the distributor, it is possible to mix and homogenize several different liquids and solutions before filling the hollow body located at the outlet opening. This is especially interesting when e.g. - Comparable with a liquid chromatography - a gradient is used to increase the specificity of the analyte-particle binding during washing.

Preferably, the mixing chamber has a stirring device. This substantially facilitates the generation of a homogeneous mixture in or at the distributor. Such mixing devices are already known for use in chromatography and can thus be used directly for the purpose of the invention. Nevertheless, it is entirely possible to dispense with a stirring device when the mixing of the liquids takes place by means of flow effects.

Tumbular hollow bodies are preferably attached to the at least one inlet opening.

These tubular hollow bodies serve to direct liquids and gases over greater distances into the distributor, whereby e.g. Hoses commonly used in chromatography are particularly well suited.

Particularly preferably, the tubular hollow body is designed as Kapilla¬ re.

The use of capillaries makes it possible to use small volumes of sample and liquid, on the one hand small amounts of sample sufficient to carry out investigations, and on the other hand miniaturization of the device according to the invention is made possible. The inner diameter of the capillaries used is a maximum of 3 mm, preferably a maximum of 2 mm, maximum lmm, maximum 0.5mm, maximum 0.3mm, maximum 0.2mm, maximum 0.1mm, maximum 500μm. Furthermore, the minimum inner diameter depends on the size or diameter of the functionalized magnetic particles used, the inner diameter should be at least twice or at least three times as large as the diameter of the particles.

Preferably, the hollow body connected to the outlet opening has at least one cross-sectional widening, in particular at least one chamber.

A cross-sectional widening of the tubular hollow body serves, for example, to form a kind of chamber, in which the mixing of the particles with solutions introduced into the device is promoted due to the currents generated by the flow and the resulting turbulences. The chambers may optionally be delimited on one side with a liquid-permeable barrier.

According to the invention, a tubular hollow body attached to an outlet opening has a plurality of cross-sectional enlargements occurring one behind the other, the hollow body being one or more pieces, i. a plurality of hollow bodies are connected to each other, is formed. A multi-part arrangement of several cross-sectional enlargements would make it possible, for example, to provide different chambers with differently functionalized particles and thus allow the analysis of several analytes simultaneously. For the parallel analysis of several samples or several different analytes in a sample, a plurality of outlet openings may alternatively be provided with tubular hollow bodies according to the invention. As a result, several hollow bodies can be addressed individually by the changeover of the distributor.

According to a preferred embodiment, the tubular hollow body connected to the outlet opening has at least partially transparent regions.

Transparent areas are particularly advantageous if an event, such as a color reaction, should be made visible within the hollow body. Also, a position control of the particles located in the hollow body would be possible with appropriate particle size or density. These ranges should, if, for example, photometric measurements are carried out directly on the hollow bodies, the light should pass through as unhindered as possible. to let. Therefore, the transparent areas may also consist of materials other than the hollow body (eg glass).

The transparent regions are preferably designed as measuring cells. Measuring cells which can be used according to the invention are already known in photometry, chromatography, etc. as flow cells and can be introduced into the flow of the device accordingly.

Preferably, the means for immobolization is a magnetic device which is arranged to be relatively movable with respect to the hollow body connected to the outlet opening of the distributor.

In order to immobilize the analyte-binding particles, which are preferably of super, para- or ferromagnetic nature, to a defined region in the interior of the hollow body, it is necessary to attach a magnetic field, which is generated by a magnetic device, to the hollow body. Preferably, this device is arranged movably, whereby the area of the immobilization can be flexibly selected. In a preferred embodiment, the magnetic device is arranged to be movable at least approximately perpendicularly to the tubular hollow body. Furthermore, such an arrangement allows the use of a permanent magnet which can be moved away from the hollow body when the magnetic field is no longer desired on the hollow body.

According to a preferred embodiment, the magnetic device comprises an electric or permanent magnet.

An electromagnet has the advantage that the magnetic field can be controlled by means of a circuit. Nevertheless, permanent magnets are also particularly suitable for use in the device according to the invention.

Preferably, a filter or a filter combination is attached to at least one inlet opening or to at least one outlet opening or to at least one tubular hollow body connected thereto.

The preferred use of filters at the inlet or outlet openings or at the tubular hollow bodies connected thereto has a number of advantages. Thus, for example, solid substances can be removed from the sample before they enter the device and interfere with the analysis. Fer¬ ner can be removed by appropriate filters and other unwanted sample components (eg leukocytes in whole blood) become. Other substances which interfere with the analysis can also be removed from the sample by the use of substance-specific filters (compare affinity column in chromatography). Thus, the sample preparation can be omitted partially or completely, whereby the use of the Vor¬ direction of the invention, for example, in emergency medicine is particularly advantageous.

According to another preferred embodiment, the filter is a plasmapheresis filter.

Particularly suitable filters for the immune platform are those whose structure is suitable for separating cell components from whole blood. Such filters are used in blood purification for dialysis machines or in lateral flow assays (for example from Schlei cher & Schuell, "AccuFlow Plus").

According to a preferred embodiment, a detector is arranged on the hollow body connected to the outlet opening of the distributor, the detector being selected from the group consisting of a photometer, fluorometer, scintillation detector, CCD detector and combinations thereof.

The arrangement of a detector on the hollow body, which is attached to the Aus¬ lassöffnung, has proved to be particularly advantageous, since it is thereby made possible to analyze the binding of an analyte of particles directly in the hollow body. It is essential in such an embodiment that the signal to be measured can penetrate the hollow body at the position of the detector. For example, the hollow body is transparent when a color reaction or fluorescence is measured. Nevertheless, the detector can also be positioned outside the device according to the invention or not on the hollow body. In this case, the analyte bound to the particles is transferred as a suspension into further vessels and subsequently determined in a separate detector.

Preferably, the device according to the invention is designed as a cassette.

The design of the device as a cassette makes it possible to flexibly combine the device with other other analyzers (eg blood gas machines). The cassette according to the invention can consist only of tubular hollow bodies (in which optionally already functionalized magnetic particles are present), which are introduced into a device which corresponds to the invention a distributor, a displacer and at least one magne¬ tables device provides. By providing corresponding connecting elements on the cassette and in the device, the cassette can be introduced by a few simple steps. Alternatively, the cassette may additionally comprise a distributor or a displacer or a magnetic device or combinations thereof.

A further aspect of the present invention relates to a method for determining at least one analyte in a sample with a device according to the invention comprising the steps:

Providing a sample,

Incubation of the sample with magnetic analyte-binding particles inside or outside the tubular hollow body connected to the outlet opening of the distributor,

optionally introducing the sample-particle suspension into the hollow body connected to the outlet opening of the distributor, if the incubation was not carried out in the hollow body,

Washing the particles to remove unbound sample components,

optionally introducing agents for detecting the analytes bound to the particles in the hollow body, and

Detecting the analytes bound to the particles, wherein the particles are immobilized on an inner boundary of the hollow body during or between the steps or durchge by a magnetic device for immobilization.

According to the invention, the magnetic particles can be immobilized on the hollow body during the entire process or only for example when changing the solutions or when washing the particles.

According to a preferred embodiment, the detection takes place inside or outside the hollow body connected to the outlet opening of the distributor.

The detection of analytes bound to magnetic particles can be carried out as described above with a wide variety of detectors, depending on which analyte is to be detected by which detection means. In this case, the detection can take place either directly in the hollow body or after the particles loaded with analyte from this hollow body into a vessel (eg in a cuvette for a photometer or a microtiter plate) was transferred.

For further characterization of the analyte, it may be separated from the particles and further analyzed, e.g. Mass spectrometry. This separation can take place, for example, by changing the pH conditions, salt concentration or by addition of a competitive substance displacing the analyte (for example analogs) (see chromatographic methods).

According to a preferred embodiment, the sample is selected from the group consisting of body fluids, in particular blood, blood serum and saliva, urine, perspiration, tissue samples, cell culture supernatants, in particular eukaryotic and prokaryotic culture supernatants, environmental analytical solutions and puncture liquids of various organs.

Due to the possibility of being able to bind analytes of various kinds to the particles according to the invention and thus to carry out a quantitative and qualitative determination, it is accordingly possible to use samples of various kinds. Clinical specimens have proven particularly suitable for carrying out the method disclosed herein, since such specimens must be examined quickly, simply and accurately in order to provide the patient with the most efficient therapy in the shortest possible time. Analytes from solid samples, e.g. Tissue samples or suspensions (cell suspensions, whole blood and the like) can be extracted from them before starting the analysis.

Extraction or separation can be carried out by suitable solvents or by homogenization and removal of the solid constituents (by centrifugation or filtration), it being possible for filtration to be carried out directly on the apparatus according to the invention or within it (eg by providing Filtering on the device according to the invention).

Preferably, the magnetic analyte-binding particles have at least one chemical modification.

By chemically modifying the particles, they can be functionalized so that the particles are able to specifically bind an analyte from a sample.

According to a preferred embodiment, the chemical mixed modification an analyte-specific binding partner.

In order to specifically bind the analyte to be examined, it is advantageous to identify a corresponding binding partner and apply it to the particles.

The chemical modification comprises a substance selected from the group consisting of proteins, in particular antibodies, antibody fragments, protein A, protein G and lectins, biotin, streptavidin, avidin, phages, phage proteins, viruses, vi¬ rale proteins / particles , Nucleic Acids, Oligonucleotides, and combinations thereof (see "Antibodies, a laboratory manual", Ed Harlow, David Lane; ColD Spring Harbor Laboratory Press, 1988).

Corresponding protocols or instructions on how binding partners can be bonded to magnetic particles according to the invention are known to those skilled in the art and are discussed in detail in the prior art (see, for example, US Pat. No. 4,628,037, EP 0180384, Schalkhammer, TGM (ed.) 2002. Analytical Biotech Basel, Boston, Berlin: Birkhäuser Verlag, Iacob G et al (2001) Rev Med Chir Soc Med Nat Iasi 105: 69-75)

According to a preferred embodiment, the means for detecting the analyte comprises a further binding partner of the analyte.

In order to detect an analyte, the use of a corresponding binding partner is particularly well suited, which if appropriate is more easily or to a greater extent detectable than the analyte to be determined.

Preferably, the further binding partner is conjugated with a marker.

The binding partner, which binds to the analyte on the particles, may be conjugated to a label. Such markers are e.g. from immunology (see ELISA, RIA and the like). Above all, a marker serves to indicate the presence of a conjugate directly or indirectly via a further reaction.

According to a preferred embodiment, the marker is an enzyme, in particular horseradish peroxidase, β-galactosidase and alkaline phosphatase, radioactive isotope, BrDU, digitonin, Ki67, PCNA, ruthenium compounds, such as Ru chelates, tris (2,2'-bipyridines) ruthenium (II) (see, for example, WO92 / 14138), or combina tions thereof.

The means for detection preferably comprise a substrate, which is reacted by the label of the conjugated binding partner, which substrate can be selected from the group consisting of luminol, dixethanes, acridine esters and combinations thereof.

The use of an enzyme marker which converts a substrate can lead to an amplification of the detection signal and thus make the detection method more sensitive.

Furthermore, the presence of an analyte on the particles can be easily determined by methods such as PCR, ELISA or PAP-APAAP.

The volume of the sample is preferably from 1 μl to 1 ml, preferably from 2 μl to 500 μl, in particular from 5 μl to 200 μl.

According to the invention, any sample volumes can be analyzed with the device disclosed herein, but preferably small volumes are used in certain fields of application (for example a blood sample in a premature infant in the clinic). The use of small volumes up to a maximum of 5 ml, a maximum of 4 ml, a maximum of 2 ml, a maximum of ImI, a maximum of 500 μl, a maximum of 200 μl in the method according to the invention is made possible above all by the provision of a corresponding device.

According to a further aspect of the present invention, the device according to the invention is used in combination with a Blut¬ gas automaton, whereby the use of a blood gas machine with respect to the determination of analytes in the blood can be dramatically expanded.

The device according to the present invention can be connected in a simple manner with the aid of capillaries, connecting pieces and valves to an outlet opening or output capillary of a blood gas automat. In this case, the device can also be designed as a disposable or reusable cassette, which can be exchangeably introduced into a blood gas automat. Automatic blood gas analyzers are sufficiently well known to the person skilled in the art, for example, by the companies Roche ("Roche COMPACT 3") and Osmetech ("Osmetech OPTI CCA Blood Gas Analyzer").

The invention will be further explained with reference to the following figure and the following example, but without being limited thereto.

1 shows an apparatus for determining an analyte which comprises a distributor 1 which has a plurality of inlet openings 2-6, an inlet opening 7 for mounting a displacer 8 (FIG. drängereingangsöffnung) and an outlet opening 9 has. At the outlet opening 9, a tubular hollow body 10 is mounted, on which a magnetic device 11 is arranged. A detector 12 for direct or indirect determination of the analyte immobilized on the magnetic particles is likewise arranged along the hollow body 10.

Via a distributor 1, by means of a displacer 8 in succession, the sample and other required reagents, such as buffers and the like, sucked and optionally in¬ with the magnetic particles in the device according to the invention in¬ cubiert. To guarantee a uniform distribution of the particles and the solutions, the resulting suspension can be mixed in a mixing chamber.

Alternatively, this suspension can be pumped into the tubular hollow body 10 of the outlet and mixed with or without forward and backward movement of the suspension.

The analyte to be determined is bound by appropriate interactions to the magnetic particles suspended in the solution. The magnetic particles are retained in the outlet hollow body 10 by applying a magnetic field to its outer boundary within the boundary. The entire volume of the suspension must be guided past the applied magnetic field by means of a displacement 8 by means of a corresponding displacement and return movement in order to quantitatively immobilize the magnetic particles.

With continued fixation of the magnetic particles, a washing process is then carried out by means of displacer 8. In this case, an amount of washing buffer solution corresponding to the assay is sucked through the distributor 1 and pumped through the outlet hollow body 10. The process can be repeated one or more times as necessary.

An optional optimization of the washing process can be achieved by pumping the corresponding wash solution volume into the outlet hollow body 10, removing the magnetic particle fixation by removing the magnetic field supplied outside the hollow body 10 and thus achieving a release of the magnetic particles into the wash solution. A subsequent movement of the newly suspended particles by gently pumping back and forth the resulting suspension by means of displacer and then immobilizing the magnetic Particulate particles by applying a magnetic field is part of a very effective washing process.

Thereafter, the magnetic particle fixing process and the general washing process described above may be repeated.

After final magnetic fixation of the particles, the excess wash buffer is removed from the outlet hollow body 10 in the next step by drawing in a quantity of gas predetermined by the respective design and subsequent ejection.

Subsequently, a necessary for complete detachment of the magnetic particles from the inner boundary of the hollow body 10 amount of substrate is sucked through the manifold 1. The substrate is pumped into the hollow body 10 and brought into contact with the fixed magnetic particles and determined by a detector 12 (PMT, diode, CCD, or the like), the re¬ present for the analyte electromagnetic radiation.

A further optimization of the quantification can be achieved by removing the magnet into the hollow body 10 after the supply of the substrate solution, whereby the previously magnetically fixed particles are released again and by gently pumping back and forth the resulting suspension by means of displacer 8 be mixed with the substrate. For the subsequent, as described above, quantification, the suspension is placed in a detection window or from the hollow body 10 in another vessel.

Finally, the entire device is prepared with wash buffer and appropriate cleaning solutions (washing) for the next measurement.

Example:

Material and methods:

An 8-channel distributor (Hamilton, type N ° 86918) with 100 μl syringe (Hamilton, type N ° 81022) and Teflon capillaries (Hamilton, type N ° 88907) connected to Luer terminals, microtiter plate white (Nunc, type N ° 437-591), StabilZyme AP (SurModics, Art-N ° SAOI-1000), Streptavidin Magnetic Particles (Aureon Biosystems, Art N ° A010101) and Dynabeads M-280 Streptavidin (Dynal, Art N ° 112.06), Biotin Peroxidase (Sciotec, 5 mg / ml), Peroxidase Substrate (Lumigen, Art-N ° PSA-100), Mediators PhL Luminometer (Aureon, Art-N ° G010001), Neodymoronate Magnet (Magnet Sales, Art N ° NIRD011SS / W), washing solution is TPBS.

For the positive control, biotin peroxidase 1: 1,000,000 was diluted with StabilZyme AP using StabilZyme AP as the negative control. The Streptavidin Magnetic Particles were also 1:40 diluted in StabilZyme AP.

First, 5 μl of streptavidin magnetic particles (particles) were sucked in via the syringe and placed in the distributor. The distributor was switched to the outlet position and the suspension was transported into a capillary which was attached to the outlet opening. A magnet was brought into the vicinity of the capillary, the magnetic particles immobilized on the inner boundary of the capillary and the liquid ejected. Thereafter, 5 μl of the positive control or the negative control were aspirated via a second inlet and switched to the outlet by switching to the capillary, the magnet was removed and the particles were brought back into suspension by reciprocating the liquid and for 10 minutes incubated. The magnet was repositioned and the particles trapped. The liquid was ejected and washed with 25 .mu.l wash solution from a third inlet with applied magnetic field ge. With another 25μl washing solution, the particles were washed with the magnet removed. Subsequently, the particles were resuspended with 25 μl substrate solution, the resulting suspension was ejected into a microtiter plate and measured in the luminometer with 1 second integration time.

Results:

The results of the measurements are shown in Table 1, which were given in relative light units (RLU).

Table 1

Claims

claims:

1. An apparatus for determining at least one analyte in a sample comprising a distributor (1) having at least one inlet opening (2-6) for introducing liquids, suspensions and / or gases into the distributor (1) and having at least one outlet opening ( 9) for removing liquids, suspensions and / or gases from the distributor (1), wherein at at least one outlet opening (9) connected to the manifold (1) tubular hollow body (10) is attached to which a magnetic ¬ direction (11) for the temporary immobilization of a Ein¬ lassöffnung (2-6) in the interior of the hollow body (10) eingebrach¬ te or inside the hollow body (10) located Magne¬ tables analytbindenden particles to an inner boundary of the hollow body (10) is arranged, characterized in that a displacer (8) is arranged on the distributor.

2. Apparatus according to claim 1, characterized in that the displacer (8) is designed as a pump or syringe.

3. Apparatus according to claim 1 or 2, characterized in that the distributor (1) is designed as a rotary valve.

4. Device according to one of claims 1 to 3, characterized gekenn¬ characterized in that the distributor (1) is designed as a multi-way valve.

5. Device according to one of claims 1 to 4, characterized gekenn¬ characterized in that the distributor (1) comprises a mixing chamber.

6. Apparatus according to claim 5, characterized in that the mixing chamber has a stirring device.

7. Device according to one of claims 1 to 6, characterized gekenn¬ characterized in that at the at least one inlet opening (2-6) tu¬ buläre hollow body are attached.

8. Device according to one of claims 1 to 7, characterized gekenn¬ characterized in that the hollow body is designed as a capillary.

9. Device according to one of claims 1 to 8, characterized gekenn¬ characterized in that connected to the outlet opening (9) Hohl¬ body (10) has at least one cross-sectional widening, insbesonde¬ re at least one chamber.

10. Device according to one of claims 1 to 9, characterized ge indicates that the hollow body (10) connected to the outlet opening (9) at least partially transparent areas auf¬.

11. Device according to one of claims 1 to 10, characterized ge indicates that the magnetic device is arranged to be movable relative to the with the outlet opening (9) of the distributor (1) connected to the hollow body (10).

12. Device according to one of claims 1 to 11, characterized ge indicates that the magnetic device (11) comprises an electromagnet.

13. Device according to one of claims 1 to 12, characterized ge indicates that the magnetic device (11) comprises a permanent magnet.

14. Device according to one of claims 1 to 13, characterized ge indicates that at least one inlet opening or at least one outlet opening or at least one tubular body connected thereto a filter or a Filterkom¬ combination is attached.

15. The device according to claim 14, characterized in that the filter is a plasmapheresis filter.

16. Device according to one of claims 1 to 15, characterized ge indicates that at the outlet opening (9) of the distributor (1) connected to the hollow body (10), a detector (12) is arranged.

17. The device according to claim 16, characterized in that the detector (12) is selected from the group consisting of a photometer, fluorometer, scintillation detector, CCD detector and combinations thereof.

18. Device according to one of claims 1 to 15, characterized ge indicates that the device is designed as a cassette.

19. A method for determining at least one analyte in a sample with a device according to one of claims 1 to 18, comprising the steps:

Providing a sample,

Incubation of the sample with magnetic analyte-binding particles inside or outside the tubular hollow body (10) connected to the outlet opening (9) of the distributor (1),

if appropriate, introduction of the sample-particle suspension into the hollow body (10) connected to the outlet opening (9) of the distributor (1), if the incubation was not carried out in the hollow body (10),

Washing the particles to remove unbound sample components,

optionally introducing at least one means for detecting the analyte bound to the particles into the hollow body (10), and

Detection of the analytes bound to the particles, the particles being immobilized during or between the steps or by applying a magnetic field to the inner boundary of the hollow body (10).

20. Method according to claim 19, characterized in that the detection takes place within the hollow body (10) connected to the outlet opening (9) of the distributor (1).

21. Method according to claim 19, characterized in that the detection takes place outside the hollow body (10) connected to the outlet opening (9) of the distributor (1).

22. The method according to any one of claims 19 to 21, characterized ge indicates that the sample selected from the group consisting of body fluids, especially blood, blood serum and spei¬ chel, urine, sweat, tissue samples, cell culture supernatants, in particular eukaryotic and prokaryotic Culture supernatants, environmental solutions and puncture liquids differ whose organs are.

23. The method according to any one of claims 19 to 22, characterized ge indicates that the magnetic analyte-binding particles have at least one chemical modification.

24. The method according to claim 23, characterized in that the chemical modification comprises an analyte-specific binding partner.

25. The method according to claim 23 or 24, characterized in that the chemical modification of a substance selected from the group consisting of proteins, in particular antibodies, Anti¬ body fragments, protein A, protein G and lectins, biotin, streptavidin, avidin, phage, phage proteins , Viruses, viral proteins / particles, nucleic acids, oligonucleotides and combinations thereof.

26. The method according to any one of claims 19 to 25, characterized ge indicates that the means for detecting the analyte comprises a further binding partner of the analyte.

27. The method according to claim 26, characterized in that the further binding partner is conjugated with a marker.

28. The method according to claim 27, characterized in that the marker is a radioactive isotope, ruthenium compounds or a fluorescent dye.

29. The method according to claim 27, characterized in that the marker is an enzyme, in particular horseradish peroxidase, ß-galactosidase and alkaline phosphatase.

30. The method according to claim 29, characterized in that a substrate is used for the detection of the marker.

31. The method according to claim 30, characterized in that the substrate is selected from the group consisting of luminol, di- ethanes, acridine esters and combinations thereof.

32. The method according to any one of claims 19 to 31, characterized ge indicates that the volume of the sample lμl to ImI, preferably 2μl to 500μl, in particular 5μl to 200μl, is.

33. Use of the device according to one of claims 1 to 18 in combination with a Blutgasautomaten.