EP2055384A1 - Device for identifying constituents in a fluid - Google Patents

Abstract

The device for determining the concentration of components in blood useful in a kit, comprises a measuring zone (3), two detection reagents for directly or indirectly interacting with the components, an opening (9) for bringing a fluid, a filter arranged between the opening and the measuring zone, a fluid inlet zone arranged between the opening and the filter, a ventilation system, a further measuring zone, and a zone for provisioning a blank value and/or a calibration value. The component to be detected or to be determined is a substance present in organisms and/or a biological molecule. The device for determining the concentration of components in blood useful in a kit, comprises a measuring zone (3), two detection reagents for directly or indirectly interacting with the components, an opening (9) for bringing a fluid, a filter arranged between the opening and the measuring zone, a fluid inlet zone arranged between the opening and the filter, a ventilation system, a further measuring zone, and a zone for provisioning a blank value and/or a calibration value. The component to be detected or to be determined is a substance present in organisms, a biological molecule, a medicinal substance and/or a protein. The presence and/or the concentration of the component in the measuring zone are determinable by luminescence, fluorescence, autofluorescence, chemiluminescence, electrochemiluminescence, spectral absorption photometry and/or bioluminescence. The filter is formed to separate solid components of the blood flowing-through the filter, subsequently to separate the solid and the liquid phase of the blood from each other and to separate serum or plasma from the blood. A pressure lying over ambient pressure is exercised on the blood. The blood is introduced through the filter into the measuring zone under the pressure. The pressure lying under the ambient pressure prevails in the device. The detection reagent is provided in the measuring zone and changes its optical characteristics during interaction. The opening has a one-way valve, and a luer-lock. The fluid inlet zone is formed and/or delimited by a flexible film. The device is a one-way-device and is compatible with commercial detection devices. The device has measures of a commercial cuvette or other commercial measuring vessels and a diameter of 10 mm and/or a length of 50+- 15 mm, or an adapter is adjusted to the size of a commercial cuvette. The ventilation system is connected with a measuring chamber by a narrow groove or a gap. The measuring zone is present in the form of a small tube with the ventilation system. The first detection reagent in the device is present above the measuring zone and is immobilized at the determined area in the measuring zone. The detection reagent is Pico-Green(RTM: Ultrasensitive fluorescent nucleic acid stain), Alexa(RTM: Superior fluorescent dyes), ethidium bromide, and SYBR(RTM: Asymmetrical cyanine dye) or Sytox(RTM: Fluorescent nucleic acid stain). Independent claims are included for: (1) a method for determining the concentration of components in blood by a device; and (2) a kit.

Description

The present invention relates to an apparatus and a method for the detection, in particular for the determination of the concentration, of constituents in blood or water. Furthermore, the present invention relates to the use of a device or a method for the determination of constituents in blood. Finally, the invention relates to a kit comprising the device and a DNA standard.

Below is a series of documents cited. The contents of these documents including operating instructions are hereby incorporated in their entirety by reference.

Medical diagnostic procedures are routinely used to estimate the clinical status of patients by determining the presence and / or concentration of solutes in the blood. So far, analyzes of the blood plasma and / or serum must be performed with a centrifugation step so that the blood plasma to be analyzed can be separated from the solid blood components. Obviously, there are no suitable alternative methods or apparatus for separating the solid blood components. Centrifugation is not only time-consuming, but also represents a major hurdle, especially in development areas, because there is often not only a shortage of suitable centrifuges but also the power to operate them. In addition, in many areas of the world, there is often no skilled person who could easily carry out blood analyzes by known means.

There is therefore a need for a device or a method which allows the analysis of substances located in blood plasma and / or serum, in particular without centrifugation. Moreover, a corresponding device or a Procedures allow even untrained personnel without time-consuming training to enable the fast, clean and correct handling of such analyzes.

In US 5,186,843 For example, a material for separating plasma or serum from blood is described. The material includes glass microfibers, cellulosic fibers and synthetic textile staple fibers. This medium is used to obtain small amounts of plasma that collect within the separation layer. However, the still necessary collection and further handling of the liquid for a quick and clean diagnosis hinder.

In US 4,816,224 describes a device for the separation of plasma or serum from whole blood, which can be designed differently. Glass fibers, which occupy a large volume, serve for separation. The device may comprise a plurality of filter layers.

In US 2006/0029923 a device is described which allows the separation of plasma or serum from solid blood components by means of a filter membrane mounted in the device and pressure. The corresponding device may include a detection strip that allows the detection of components in the blood by immunochemical detection methods. However, these strips do not allow direct detection without further steps to be carried out by the staff and without the use of further auxiliaries or auxiliaries. The device is primarily intended for obtaining blood plasma or serum for further study.

Thus, devices as described above are primarily used to provide small amounts of plasma or serum for further analysis.

With existing methods and devices for the examination of plasma or serum and / or other body fluids, which are designed to quickly obtain a measurement result, often only indicative values can be obtained, and often only statements about the presence or absence of constituents of a liquid to be measured be made. So give For example, test strips only provide information on whether or not a substance is present within the detection capacity. However, in the context of the diagnosis of certain diseases and / or conditions in samples from patients, these methods or devices are often not suitable because only accurate measurements of the concentration or content of a substance in a fluid, eg, blood, provide information about the exact condition of the patient and appropriate treatment methods.

It is therefore an object underlying the invention to provide an apparatus and a method which overcomes the disadvantages of the prior art. In particular, it is an object to provide a device by means of which the separation of plasma or serum from whole blood and an analysis of serum or plasma substances can be carried out without centrifugation steps or similar laboratory processing steps of the recovered serum or plasma are necessary. Further or additional objects of the present invention are the provision of a simple, safe and reliable device or method which allows, in particular, laypersons or non-medically trained personnel to provide a device which is simple and inexpensive to manufacture and / or store, and the provision of a corresponding kit.

This object is achieved by the features defined in the claims.

The present invention preferably relates to a device for detecting, in particular for determining the concentration, of constituents in blood, having a measuring range, a filter and at least one detection reagent for interacting with the constituents.

In the case of a measurement of autofluorescence or absorption of fluid constituents, it is also possible to dispense with a detection reagent.

By "detection" of, for example, substances is meant in particular the determination of the presence or absence of a substance. The detection limit of the measuring method determines the result within the scope of the desired accuracy.

The "concentration" of substances, especially in contrast to the absolute amount of substance, volume-independent.

In the context of the present invention, "constituents in blood" is understood to mean, in particular, substances present in blood and / or dissolved substances. The substances may in particular be organic or inorganic or a mixture of both. In principle, components of other fluids can also be determined with the device of the present invention. Fluids are to be understood as liquids with solid constituents or suspensions. Preferably, the fluid is a body fluid. Body fluids include, for example, blood, cerebrospinal fluid, urine, serous fluids, saliva, sperm, or abnormally altered stool.
Preferably, the fluid is water, in particular from lines, streams or lakes, etc. Here it can be determined, for example, whether a measured DNA correlates with the load on the water by microorganisms.

A "measuring range" in the sense of the present invention is in particular a space, preferably with a defined or definable volume, in which at least one constituent of a fluid is determined. It is preferably at least partially transparent and particularly suitable for the determination of the substance or the constituent with preferably optical methods.

For use with preferred detection reagents, which are excited, for example, by light of certain wavelength ranges and emit light of certain wavelength ranges, the measuring range is preferably for z. B. visible light and the light of the emission wavelengths permeable.

The filter in the present invention may include or include any material suitable for separation of blood or other solid-component fluids such exist. The filter may comprise, for example, polyethylene terephthalate (sold, for example, by Sekisui in a serum recovery product) or BTSSP (sold by Pall).

Already known suitable filters and / or filter materials are for. In US 4,816,224 . US 5,186,843 or US 2006/0029923 described.

Preferably, the filter comprises glass fibers but is not completely made of glass fibers. Even more preferably, the volume or weight fraction of glass fibers is between 0% and 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or between about 5% and 50% % of the filter, preferably between about 10% and 50%. Most preferred is a filter material without glass fibers. For filtering blood, the filter material should not cause hemolysis or bind analytes.

In the context of the present invention, the term "detection reagent" is used in particular to describe a substance with which the presence and / or concentration of another substance, preferably one in a fluid, eg. B. blood, plasma or water substance, detected or detected, understood. In this case, a detection reagent preferably has the property under certain conditions to enable or condition a detectable reaction. A detection reagent preferably interacts directly with the substance to be determined. It is either a covalent or a non-covalent bond with this substance. Preferably, the detection reagent fluoresces reinforced only by binding to the analyte.

The device according to the invention is preferably a ready-to-use or ready-to-use device which makes it possible to detect and in particular determine the concentration of the constituents in a simple and reliable manner without extensive preparatory measures. The present invention proves to be particularly advantageous in that a device is provided which is small, can be read with commercially available devices and / or the separation of the solid constituents of fluids, preferably from Blood, with the simultaneous measurement of components in the liquid phase connects. In this way, not only the centrifugation step is saved, which was previously necessary, for example, to separate solid blood components from the serum or plasma, but it is possible by simple and safe handling even untrained personnel, the analysis of fluids in the context of diagnostic procedures. Another advantage is the now possible accurate determination of the concentration of components, for example, to estimate the condition of patients after surgery or in certain disease states. Finally, the present device allows for the instantaneous measurement of the constituents of interest (ready-to-use) without further treatment and / or delay or other necessary measurement steps.

Preferably, the device has an opening for introducing a fluid, wherein the filter between the opening and measuring range is arranged.

Preferably, the device further comprises a fluid input region, which is preferably arranged between the opening and the measuring area, more preferably between the opening and the filter.

In the following, the area between the opening and the measuring area and / or the fluid channel, preferably comprising the fluid input area, is also summarized under the term filter area.

The filter area preferably has a capacity of between about 200 and about 2000 μl. Depending on the fluid or, preferably, the amount and texture of the blood, up to 200 μl of plasma or serum can be obtained for measurement in the measuring range. The device according to the invention preferably allows the provision of a volume to be measured in the measurement range between about 15 μl and about 80 μl, preferably between about 20 μl and about 60 μl and most preferably about 40 μl serum or plasma.

In the context of the present invention, it is preferable to determine constituents in serum or plasma or blood serum or blood plasma.

"Plasma" is preferably understood to mean, in particular, the liquid phase of the blood which is separate from solid constituents such as cells (erythrocytes, white blood cells, etc.) and which can still coagulate.

"Serum" is preferably understood to mean, in particular, the liquid fraction of the blood which is obtained after coagulation of the blood by separation of the cellular constituents mixed with platelets and coagulation factors to form the blood cake.

In principle, within the scope of the present invention, each substance can be determined using suitable detection reagents. Preferably, the ingredient to be detected or determined is a substance found in organisms. Organisms occurring substances may be organic or inorganic nature. Inorganic are, for example, minerals or mineral salts, trace elements, inorganic ions, metals and heavy metals, etc.

Organic substances can belong to different substance classes. A group of substances form proteins, including enzymes. Enzymes convert their substrate into an end product. In the context of the present invention, preferably both the enzyme and the substrate and / or the end product can be determined. Also, non-enzyme proteins may preferably be detected or determined by the apparatus of the present invention. Other groups of organic substances include vitamins and coenzymes, nucleic acids, cytokines, hormones, histones, peptides, sugars, etc.

Another group of substances are nucleic acids comprising DNA and RNA in their single and / or double-stranded forms.

Preferably, the component to be detected or determined is a biological molecule selected from the group comprising DNA, RNA, proteins, hormones, cytokines. The latter substances may be free or associated be with other proteins. For example, DNA in plasma or serum may also occur as a complex with histones and / or elastase as well as microsatellites. Furthermore, the component to be detected or to be determined is preferably the DNA / RNA of intact or intact bacteria, viruses and / or parasites from water samples contaminated with other particles. In this case, particles are retained by the filtration, while the nucleic acid contained in microorganisms can be made accessible to a measurement by suitable fluorescent dyes.

Preferably, the ingredient to be detected or determined is a drug. Drugs are substances which can be administered to an individual for controlling a pathological condition or disease. Drugs may also be the abovementioned biological molecules.

Most preferably, DNA is determined or measured. The filter used in the present invention preferably binds little or no DNA. The bound fraction is preferably less than 30%, preferably less than 20%, most preferably less than 10% of the DNA contained in the fluid or blood.

Qualitatively and quantitatively, DNA can be detected in many ways. These include PCR as well as detection by detectable agents interacting specifically with DNA. The present invention preferably permits the determination of non-cell-bound DNA. Non-cell-bound DNA can be determined by different methods. In addition to the measurement of the fluorescence after addition of an intercalating dye, the measurement of the UV absorption of DNA is also used to determine the concentration. The sample volumes required for this have now reached the low μl range (eg measurement by NanoDrop in the range from 2 μl). The lower measurement limit for DNA is z. At about 10 ng / ml for the fluorescence method.

After major operations, especially with heart-lung machine, but also after accidents with polytrauma, sepsis, burns and after ischemia / reperfusion disease (after arterial occlusion) there are strong, sometimes over-activations of the immune system. Exemplary, but not exclusive, disease states are surgery, polytrauma, soft tissue trauma, ischemia / reperfusion disease, infarct, ischemia, embolism, infection, sepsis, transplantation, intoxication, eclampsia, drug side effects or transfusions. These can cause temporary or permanent damage in organs, but can also lead to death. The cellular component of this immune response is mediated by neutrophil granulocytes. To assess the consequences of activation, it is important to be up-to-date about the extent of the event.

As in US 60 / 827,571 discloses that the concentration of non-cell-bound DNA released from granulocytes in the blood increases in the presence of certain pathological events that are not caused by cancer or pathogens. Depending on the pathological state, this non-cell-bound DNA could be free and / or in the form of so-called NETs, DNA networks decorated with proteins, which in this case are not only produced for the defense against microorganisms, but also generally released in the course of immune responses that are caused by certain pathological events. While parts of these NETs stick to the capillaries, another part breaks off as a result of the blood flow and is in the plasma and serum.

The present invention provides an apparatus and method for detecting the NETs that are already released within minutes of activation of the granulocytes producing them. Proofing in patient samples can lead to timely responses from the attending physicians who can save lives.

More free DNA in the blood is released from dead cells. These cells may be dead by apoptotic or necrotic events. In the course of some therapies, for example of cancer with chemo- or Radiotherapy induces apoptosis of cells, including blood cells. The therapeutic success can thus be detected directly and finely graded with the present invention.

Preferably, the presence and / or concentration of the component in the measuring range, preferably depending on the detection reagent used, by means of luminescence, fluorescence, chemiluminescence, electrochemiluminescence, spectral absorption photometry, autofluorescence and / or bioluminescence determinable.

Preferably, the filter is adapted to separate solid components of the fluid flowing through the filter, and in particular to separate the solid and liquid phases of the fluid from each other. Some filter materials can be very brittle or fragile, so stabilization is necessary. Depending on the nature of the filter, it may contain an element for stabilization. Stabilizing elements are preferably stabilizing frames or frames or superimposed or integrated networks of a stable material, such as metal or plastic.

Preferably, the fluid input region, which is preferably encompassed by the filter region, of the device is designed such that an above ambient pressure is exerted on the fluid. The device is preferably designed such that such a pressure during the introduction of the fluid, preferably by a syringe or the like, is constructed.

Preferably, the device is designed such that the fluid is introduced under pressure through the filter in the measuring range. This pressure can preferably be exercised manually, for. B. by the piston of a syringe containing the unfiltered fluid to be measured.

According to a preferred embodiment of the invention prevails in the device is a pressure lying below the ambient pressure, preferably a vacuum. If the sample to be determined is brought into contact with the device, causes the low pressure or the vacuum that the sample is passed or sucked without externally applied force in the device and inside the device, the solid components are separated from the fluid. In this case, the measuring range is preferably made of a preferably elastic material, which endeavors to assume a defined position or shape. Preferably, the device is made of elastic material that is deformed such that the inner volume of the measuring chamber is smaller than that of the defined position, so that it unfolds eg when introducing the sample or the sample is sucked in during deployment. Further preferably, the measurement region is made of an inelastic material, wherein the region within the measurement region has a lower pressure than the ambient pressure before the sample enters the device.

In a further preferred embodiment, the detection reagent is provided in the measuring area or in a cavity / lumen located just before the measuring area, which preferably extends between the filter and the measuring area.

In a further preferred embodiment, the detection reagent is provided in the filter or in spatial proximity to the filter.

In a further preferred embodiment, the detection reagent interacts directly or indirectly with the constituent to be determined. Direct interaction is preferably in the case of the determination of DNA with reagents that attach to the DNA. In this case, the attachment can be covalent or noncovalent.
Certain substances integrate into double-stranded and single-stranded DNA without covalent binding with the DNA (intercalators). In part, the substances only gain the property of fluorescence. By accumulation in the DNA and irradiation with light of certain wavelengths, these substances emit light of different wavelengths, which is quantitatively measurable and correlated with the amount of DNA. Fluorescent dyes may also be other than intercalators and be contacted by chemical modification with the DNA to be measured. In a further preferred embodiment, the detection reagent is selected from the group comprising Pico-Green ™, Alexa or Sytox® dyes, ethidium bromide and SYBR® dyes.

In the context of the present invention, preferably a quantitative determination of a substance in the measuring range is carried out. For this purpose, if only one detection reagent is present, it is advantageous for the detection reagent, when interacting with the target molecule, to change its properties such that it becomes detectable. As described above, intercalators become detectable only upon incorporation into nucleic acids, whereas in the unbound state they do not exhibit this property. The detection reagent used in the present invention may already be detectable prior to interaction with the target molecule, but preferably alters its pertinent properties upon interaction with the target molecule. This is preferably done by shifting the absorption maximum or the emission wavelength of a detection reagent.

The measurement of the non-cell-bound DNA preferably comprises the determination of the fluorescence emission after addition of a fluorescent dye to the plasma or serum.

In principle, all fluorophores can be used within the scope of the invention. The following non-exhaustive list gives examples of suitable fluorophores: 1.5 IAEDANS, 1,8-ANS, 4-methylumbelliferone, 4 ', 6-diamidino-2-phenylindole, 5- (and-6) -carboxy-2' , 7'-dichlorofluorescein pH 9.0, 5-carboxy-2,7-dichlorofluorescein, 5 carboxyfluorescein (5-FAM), 5-carboxynapthofluorescein (pH 10), 5-carboxytetramethylrhodamine (5-TAMRA), 5-FAM (5-carboxyfluorescein ), 5-FAM pH 9.0, 5-HAT (hydroxy tryptamine), 5-hydroxy tryptamine (HAT), 5-ROX (5-carboxy-X-rhodamine, triethylammonium salt), 5-ROX (carboxy-X-rhodamine) , 5-ROX pH 7.0, 5-TAMRA (5-carboxytetramethylrhodamine), 6 JOE, 6-carboxyrhodamine 6G, 6-carboxyrhodamine 6G pH 7.0, 6-carboxyrhodamine 6G hydrochloride, 6-CR 6G, 6-HEX, SE pH 9.0, 6-JOE, 6-TET, SE pH 9.0, 7-AAD (7-amino-actinomycin D), 7-amino-4-methylcoumarin, 7-aminoactinomycin D (7-AAD), 7-hydroxy-4-methylcoumarin, 9-Amino-6-chloro-2-methoxyacridine, ABQ, Acid Fuchsin, ACMA, ACMA (9-amino-6-chloro-2-methoxyacridine), acridine homodimer, acridine orange, acridine orange + DNA, acridine orange, DNA & RNA, acridine red, acridine yellow, acriflavine, acriflavine Feulgen SITSA, aequorin, AFPs, Alexa 405 , Alexa 430, Alexa 488, Alexa 546, Alexa 568, Alexa 594, Alexa Fluor 350 ™, Alexa Fluor 430 Antibody Conjugate pH 7.2, Alexa Fluor 430 ™, Alexa Fluor 488 Antibody Conjugate pH 8.0, Alexa Fluor 488 Hydrazide Water, Alexa Fluor 488 ™, Alexa Fluor 532 ™, Alexa Fluor 546 ™, Alexa Fluor 568 Antibody Conjugate pH 7.2, Alexa Fluor 568 ™, Alexa Fluor 594 ™, Alexa Fluor 610 R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 647 R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 633 ™, Alexa Fluor 647 ™, Alexa Fluor 660 ™, Alexa Fluor 680 ™, Alexa Fluor 700, Alexa Fluor 750, Alizarin Complexone, Alizarin Red, Allophycocyanin (APC), AMC, AMCA-S, AMCA (Aminomethyl Coumarin), AMCA -X, amino actinomycin D, aminocoumarin, aminomethyl coumarin (AMCA), aniline blue, anthrocyl esters arat, APC (allophycocyanin), APC-Cy7, APTRA-BTC, APTS, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon, Yellow 7 GLL, Atabrin, ATTO-TAG ™ CBQCA, ATTO-TAG ™ FQ, Auramine, aurophosphine, aurophosphin G, BAO 9 (bisaminophenyloxadiazole), BCECF (high pH), BCECF (low pH), BCECF pH 5.5, BCECF pH 9.0, berberine sulfate, beta lactamase, BFP, BFP / GFP FRET, bimane, bis-acridine orange , Bisbenzamide, bisbenzimide (Hoechst), bis-BTC, Blancophor FFG, Blancophor SV, BOBO-3 DNA, BOBO ^™ -1, BOBO ^™ -3, Bodipy 492/515, Bodipy 493/503, Bodipy 500/510, Bodipy 505/515, Bodipy 530/550, Bodipy 542/563, Bodipy 558/568, Bodipy 564/570, Bodipy 576/589, Bodipy 581/591, Bodipy 630/650-X, Bodipy 650/665-X, Bodipy 665 / 676, Bodipy FI, Bodipy FL ATP, BODIPY FL conjugate, BODIPY FL, MeOH, Bodipy FI-Ceramide, Bodipy R6G SE, Bodipy TMR, Bodipy TMR-X Conjugate, Bodipy TMR-X, SE, Bodipy TR, Bodipy TR ATP BODIPY TR-X Phallacidin pH 7.0, Bodipy TR-X SE, BODIPY TR-X, MeOH, BODIPY TR-X, SE, BOPRO-3, BO-PRO-3 DNA, BO-PRO ^™ -1, BO-PRO ^™ -3, Brilliant Sulfoflavin FF, BTC, BTC-5N, Calcein, Calcein Blue, Calcein pH 9.0, Calcein / Ethidium Homodimer, Calcium Crimson, Calcium Crimson Ca2 +, Calcium Crimson ^™ , Calcium Green, Calcium Green-1, Calcium Green-1 Ca2 +, Calcium Green-2, Calcium Green-5N, Calcium Green-C18, Calcium Orange, Calcofluor White, Carboxy-X-rhodamine (5-ROX), Cascade Blue, Cascade Blue BSA pH 7.0, Cascade Blue ^™ , Cascade Yellow, Cascade Yellow antibody conjugate pH 8.0, Catecholamines, CCF2 (Gene Blazer), CFDA, CFP, CFP (Cyan Fluorescent Protein), CFP / YFP FRET, Chlorophyll, Chromomycin A, Chromomycin A, CI NERF pH 2.5, CI NERF pH 2.5, CI NERF pH 6.0, CI -NERF pH 6.0, CL-NERF, CMFDA, coelenterazine, coelenterazine cp, coelenterazine f, coelenterazine fcp, coelenterazine h, coelenterazine hcp, coelenterazine ip, coelenterazine n, coelenterazine O, coumarin phalloidin, C-phycocyanines, CPM, CTC, CTC formazan , Cy2 ™, Cy3.1 8, Cy3.5 ™, Cy3 ™, Cy5.1 8, Cy5.5 ™, Cy5 ™, Cy7 ™, Cyan 3, Cyan 6 "cyan GFP, cyclic AMP fluorosensor (FiCRhR), CyQuant , CyQUANT GR-DNA, Dabcyl, Dansyl, Dansylamine, Dansylcadaverine, Dansylchloride, Dansyl DHPE, Dansylfluoride, DAPI, Dapoxyl, Dapoxyl 2, Dapoxyl 3, DCFDA, DCFH (Dichlorodihydrofluorescein diacetate), DDAO, DHR (Dihydorhodamine 123), Di-4- ANEPPS, Di-8 ANEPPS, Di-8-ANEPPS (non-ratio), Di-8-ANEPPS-lipid, DiA (4-Di-16-ASP), Dichlorodihydrofluorescein diacetate (DCFH), DiD, DIDS, Dihydorhodamine 123 (DHR ), Dihydroethidium, DiI (DiIC18 (3)), di IC ₁₈ ("DiD") dinitrophenol, DiO (DiOC18 (3)), DiR, DiR (DiIC18 (7)), DM-NERF (high pH), DM-NERF pH 4.0, DM-NERF pH 7.0, DNP, dopamine , DsRed, Draq5, DTAF, DY-630-NHS, DY-635-NHS, EBFP, ECFP, eCFP (Enhanced Cyan Fluorescent Protein), EGFP, Enhanced Green Fluorescent Protein (eGFP), ELF 97, Eosin, Eosin Antibody Conjugate pH 8.0 , Erythrosine, Erythrosine ITC, Ethidium Bromide, Ethidium Monoazide, Ethidium Homodimer, Ethidium Homodimer -1 (EthD-1), Ethidium Homodimer 1 DNA, Ethidium Homodimer-2, Euchrysine, EukoLight, Europium (III) Chloride, EYFP, eYFP (Enhanced Yellow Fluorescent Protein), Fast Blue, FDA, Feulgen (Pararosaniline), FIF (Formaldehyde Induced Fluorescence), FITC, FITC Antibody, FITC Antibody Conjugate pH 8.0, Flazo Orange, Fluo-3, Fluo-3 Ca2 +, Fluo-4, Fluorescein, fluorescein (FITC), fluorescein 0.1 M NaOH, fluorescein antibody conjugate pH 8.0, fluorescein indextran pH 8.0, fluorescein diacetate, fluorescein pH 9.0, fluoro-emerald, fluoro-gold (hydroxystilbamidine), fluoro-ruby, fluorine X, FM 1-43, FM 1-43 lipid, FM 1-43 ™, FM 4-64, Fura Red Ca2 +, Fura Red, high Ca, Fura Red, low Ca, Fura Red ^™ (high pH), Fura Red ^™ / Fluo-3, Fura-2, high calcium, Fura-2, low calcium, Fura-2 / BCECF, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gene Blazer (CCF2), GFP (S65T), GFP red shifted (rsGFP), GFP wild type, non-UV excitation (wtGFP), GFP wild type, UV excitation (wtGFP), GFPuv, Gloxalic Acid, Granular Blue, Haematoporphyrin, HcRed, Hexidium iodide, Hoechst 33258 (bisbenzimid), Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, hydroxycoumarin, hydroxystilbamidine, hydroxystilbamidine (FluoroGold), hydroxytryptamine, indo-1, high calcium, indo-1, low calcium, indodicarbocyanine (DiD), indotricarbocyanine (DiR), Intrawhite, Cf, JC-1, JO-JO-1, JO-PRO-1, LaserPro, Laurodan, LDS 751, LDS 751 (DNA), LDS 751 (RNA), Leucophor PAF Leucophor SF, Leucophore WS, Lissamine Rhodamine, Lissamine Rhodamine B, LIVE / DEAD Kit Animal Cells, LOLO-1, LO-PRO-1, Lucifer Yellow, Lucifer Yellow, Lyso Tracker Blue, Lyso Tracker Blue-White, Lyso Tracker Green, Lyso Tracker Red, Lyso Tracker Yellow, LysoSensor Blue, LysoSensor Green, LysoSensor Green pH 5.0, LysoSensor Yellow pH 3.0, LysoSensor Yellow / Blue, Mag Green, Magdala Red (Phloxin B), Mag-Fura Red, Mag Fura-2, Mag-Fura-5, Mag-Indo-1, Magnesium Green, Magnesium Green Mg2 +, Magnesium Orange, Malachite Green, Marina Blue, Maxilon Brilliant Flavin 0 GFF, Maxilon Brilliant Flavin 8 GFF, Merocyanine, Methoxycoumarin, MitoTracker Green, Mitotracker Green FM, MitoTracker Green FM, MeOH, Mitotracker Orange, MitoTracker Red, MitoTracker Red, MeOH, Mitramycin, Monobromobimane, Monobromobimane (mBBr-GSH), Monochlorobimane, MPS (Methyl Green Pyronine Stilbene) , MRFP, NBD, NBD amine, NBD-X, NBD-X, MeOH, NeuroTrace 500/525, green fluorescent Nissl stain RNA, Nile Blue, EtOH, Nile Red, Nile Red Lipid, Nissl, Nitrobenzoxadidol, Norepinephrine, Nuclear Fast Red , Nuclear Yellow, Nylosan Brilliant Lavin E8G, OliGreen®, Oregon Green, Oregon Green 488, Oregon Green 488 antibody conjugate pH 8.0, Oregon Green 488-X, Oregon Green 514, Oregon Green 514 antibody conjugate pH 8.0, Oregon Green ^™ , Oregon Green ^™ 488, Oregon Green ^™ 500, Oregon Green ^™ 514, Pacific Blue, Pacific Blue Antibody Conjugate pH 8.0, Pararosaniline (Feulgen), PBFI, PE-Cy5, PE-Cy7, PerCP, PerCP-Cy5.5, PE-TexasRed [ Red 613], Phloxine B (Magdala Red), Phorwite AR, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, PhotoResist, Phycoerythrin B [PE], Phycoerythrin R [PE], PicoGreen dsDNA quantitation reagent, PKH26 (Sigma), PKH67, PMIA, Pontochrome Blue Black, POPO-1, POPO-1 DNA, POPO-3, POPO ™ 3-iodide, PO-PRO-1, PO-PRO-1 DNA, PO-PRO-3, Primulin, Procion Yellow, Propidium Iodide (PI), Propidium Iodide DNA, PyMPO, Pyrene, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, QSY 7, Quinacrine Mustard, Red 613 [PE-Texas Red], Resorufin, RH 414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 110 pH 7.0, Rhodamine 123, Rhodamine 123, MeOH, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B extra, Rhodamine BB, Rhodamine BG, Rhodamine Green, Rhodamine Phallicidin, Rhodamine Phalloidin, Rhodamine Red, Rhodamine WT, Rhodamine Green pH 7.0, Rhodol Green POPO-1 DNA pH 8.0, Ribogreen, Rose Bengal, R-phycocyanin, R-phycoerythrin (PE), R-Phycoerythrin pH 7.5, RsGFP, S65A, S65C, S65L, S65T, Sapphire GFP, SBFI, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, sgBFP ^™ , sgBFP ^™ (super glow BFP), sgGFP ^™ (super glow GFP), SITS, SITS (Primuline), SITS (Isothiosulphonic Acid Style Level), SNAFL calcein, SNAFL-1, SNAFL-2, SNARF calcein, SNARF1, Sodium Green, Sodium Green Na +, Spectrum Red, Spectrum Aqua, Spectrum Green, Spectrum Orange, SPQ (6-methoxy-N- (3- sulfopropyl) quinolinium), stilbenes, sulphorhodamine B can C, sulphorhodamine G Extra, SYBR® Green, SYBR® Green I, SYBR® Green II, SYBR® Gold, SYBR® Safe DNA, SYPR O Ruby, SYTO 11, SYTO 12, SYTO 13, SYTO 13 DNA, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 45-DNA, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Blue DNA, SYTOX Green, SYTOX Orange, Tetracycline, Tetramethyl Rhodamine (TRITC), Texas Red ^™ , Texas Red-X POPO-1 DNA pH 7.2, Texas Red-X ™ conjugate, thiadicarbocyanine (DiSC3), thiazine red R, thiazole orange, thioflavin 5, thioflavine S, thioflavin TCN, thiolyte, Tinopol CBS (Calcofluor White), TMR, TO-PRO®-1 iodide, TO-PRO®-3 iodide, TO -PRO-1, TO-PRO-1 DNA, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-1 DNA, TOTO-3, TriColor (PE-Cy5), TRITC TetramethylRodaminium ToothioCyanate, True Blue, TruRed, Ultralite, Uranin B, Uvitex SFC, wt GFP, WW 781, X-Rhod-1 Ca 2+, X-Rhodamine, XRITC, Xylene Orange, Y66F, Y66H, Y66W, Yellow GFP, YFP, YO-PRO®-1 iodide, YO-PRO-1, YO-PRO-1 DNA, YO-PRO-3, YOYO-1, YOYO-1 DNA, YOYO-3

Particularly preferred here are 4 ', 6-diamidino-2-phenylindole, 7-AAD (7-aminoActinomycin D), acridine orange, DNA & RNA, acridine red, Alexa Fluor 594 ™, Alexa Fluor 610 R-Phycoerythrin streptavidin pH 7.2, Alexa Fluor 647 R-Phycoerythrin Streptavidin pH 7.2, Alexa Fluor 633 ™, Alexa Fluor 647 ™, Alexa Fluor 660 ™, Alexa Fluor 680 ™, Alexa Fluor 700, Alexa Fluor 750, allophycocyanin (APC), BOBO-3 DNA, BOBO ^™ -3, Bodipy 650/665-X, Cy5.5 ^™ , Cy5 ^™ , DAPI, DDAO, Draq5, ethidium bromide , Ethidium monoazide, ethidium homodimer, ethidium homodimer-1 (EthD-1), ethidium homodimer-1 DNA, ethidium homodimer-2, Hoechst 33258, Hoechst 33342, LDS 751, LDS 751 (DNA), LOLO-1, MitoTracker Red, Nile Blue-EtOH, OliGreen®, PicoGreen dsDNA quantitation reagent, POPO-1 DNA, PO-PRO-1 DNA, propidium iodide (PI), propidium iodide DNA, Ribogreen, SYBR® Green I, SYBR® Green II, SYBR® Gold , SYBR® Safe DNA, SYPRO Ruby, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTOX Orange, Texas Red ^™ , TO-PRO-1 DNA, TO-PRO-3, TO-PRO-5 , TOTO-1 DNA, TOTO-3, YO-PRO-1 DNA, YO-PRO-3, YOYO-1, YOYO-1 DNA and YOYO-3.

Most preferred is Pico-Green ™ and / or SYTOX Green.

The dose of Pico-Green ™ is preferably about 0.01 to 5 μl of reagent, preferably 0.05 to 2 μl, more preferably 0.1 to 0.5 μl of reagent, most preferably 0.15 to 0.3 μl Reagent per measurement at approx. 40 μl sample volume. The amount of reagent used is adjusted to the sample volume. In the presence of the detection reagent in solid form, the amount of solid in its content of detection reagent corresponds to the content of dissolved solid in o.g. Volumes.

Preferably, the opening of the device has a one-way valve.
Further preferably, the opening has a Luer lock. Further preferably, the filter area is formed or delimited by a, preferably elastic, film. The film is preferably made of an artificial or natural polymer or mixed polymer.

In a further preferred embodiment, the device is a disposable device.

Preferably, the filter is designed to separate serum and / or plasma from the blood. In this case, preferably no lysis of the blood cells takes place, which can influence the measurement result.

Within the filter, there may be substances that can remove (bind) bilirubin, hemoglobin or dispersed lipids (also micelles). Such substances may be, for example, titanium dioxides or colestyramine.

In the determination of DNA residual amounts of platelets may be present without adversely affecting the measurement itself. For measurements of other components, a complete separation of the platelets is advantageous, which can be done with a filter sandwich consisting of several layers of different filters at the appropriate location in the device.

Preferably, the device, in particular with respect to their dimensions, compatible with commercially available detection devices. These include, in particular, commercially available photometers, such as, for example, the Picofluor, TBS380 (Turner Biosystems, USA) or the qubit (Invitrogen, USA).

Preferably, the device and / or at least portions of the device to the dimensions of a commercially available cuvette and preferably has a diameter of about 10mm and / or a length of about 50 mm +/- 15 mm or by means of an adapter to the size of a commercially available cuvette customized.

Also preferably, the device, in particular for use with the qubit device, a cuvette in the form of a PCR tube (preferably with a use volume of 0.5 ml) on.

Also preferably, the cuvette may be miniaturized, z. B. from a clear tube made of plastic or glass, with a filling volume of about 20-100 ul, a length of about 5-25mm and an inner diameter of about 1-4 mm.

Preferably, the device has at least one venting device. The venting takes place preferably by means of a narrow groove, a channel or a gap, preferably of a few mm length, which (r) opens into the measuring chamber or from this and the other end via a membrane of the outside air or environment is disconnected. This membrane is preferably a semi-permeable membrane which is permeable to air or gas but not aqueous fluid. In the case of using a cuvette, the semipermeable membrane is particularly preferably attached to the lower end of the tube, preferably as a closure of the tube.

The present apparatus provides an apparatus having an integrated automatic size measurement chamber. In this case, the measuring range preferably connects in the filling direction or flow direction of the fluid behind the filter and fills due to the design, arrangement and vent preferably free of air bubbles with filtrate or filtered fluid. This is preferably done in a precisely determinable and correct amount, suitable or matched to the amount of Detektionsreagenz so that after filling to the semipermeable membrane no further flow can take place. The volume is determined by the volumes of the fluid spaces in the cuvette part. The present invention thus allows, in particular, an automatic quantity measurement or quantity predetermination with the advantage that in particular pipetting or other fluid handling is not necessary.

The measuring region can also already be partially prefilled, wherein this prefilling can have a dilution buffer with a detection reagent. The remaining fillable volume of the measuring range can then be filled with a quantity of filtered fluid limited by the residual volume and thus defined and can be measured after a short incubation time in a fluorescence photometer. After balancing the measured value thus obtained with a standard taking into account one blank value (or blanks), the amount of a substance that is contained in the fluid can be determined.

In a further preferred embodiment, the venting device is connected to the measuring chamber by a narrow groove or a gap, which is arranged opposite to the inlet opening of the measuring chamber.

Preferably, the measuring range is in the form of a conventional (PCR) tube, more preferably with a ventilation device, before or is designed as such, on which the remaining device is attached.

The device preferably has at least one second measuring range. This enables the simultaneous determination of several measured values or the simultaneous determination of one or more calibration values. Preferably, the device has at least one further measuring range for providing a blank or calibration value.

Preferably, the component to be determined interacts with two detection reagents. In this case, the detection or the concentration determination is carried out with two detection reagents in a two-step reaction. The first binding detection reagent interacts specifically with the constituent to be determined. This first reagent is preferably coupled to a detectable substance. The detectable substance preferably corresponds to one of the above-mentioned fluorescent dyes.

Preferably, the second detection reagent is immobilized to a certain area in the measurement area.

The first detection reagent is preferably located in the device in an area in front of the measuring area, for example in a fluid channel, filter area or in a mixing chamber leading thereto. Before entering the measuring range, the blood or the separated plasma or serum should come into contact with the first detection reagent, so that the latter can bind to the substance to be determined. The complex of the substance to be determined and the first detection reagent then passes with the plasma or serum into the measurement area, where it binds to a second detection reagent which is in a certain range of the measurement range is immobilized. By accumulating the complex at this point, the constituent to be determined becomes detectable. Excess first detection reagent remains evenly distributed in serum or plasma. A control value can be determined which reflects the concentration of the first detection reagent distributed throughout the solution. This control value can also be programmed as a threshold or limit value into a suitable measuring instrument via software. In the case of this two-step reaction, both reagents added for the detection are regarded as detection reagents, whereby the first detection reagent does not have to change its property of detectability upon binding of the constituent to be determined, as described above. However, it is preferred that the first detection reagent has this property. Both detection reagents bind directly to the component to be determined, so that the actual detection or determination of the component by detection of the first detection reagent, while the second detection reagent fixes the antigen at a position and this position after enrichment as a result of a finite flow with antigen-containing Liquid is supplied to a photo-optical measurement.

In this two-step reaction, the detection reagents are preferably antibodies or antibody fragments. In the case of the first detection reagent, the antibody is coupled to a substance to be detected. For example, R-phycoerythrin (PE), fluorescein isothiocyanate (FITC), PE-Cy5 allophycocyanin (APC), PE-Texas Red ^™ , Peridinin Chlorophyll Protein (PerCP), PerCP-Cy5.5, APC-Cy7, and / or Texas Red ^™ , etc.

Components to be determined in a two-step reaction are, for example, endogenous substances such as interleukin 6 (IL-6), hemoglobin, bilirubin, CRP, lactoferrin, procalcitonin, AT-III, protein C, p24 (HIV) or antibodies. Even foreign substances such as viruses, bacteria, drugs, toxins, drugs or fragments of the substances mentioned can thus be determined.

The device according to the invention thus enables the qualitative and / or quantitative determination of a blood component, in particular without further, manual or laboratory work steps.

The present invention also relates to a method for detecting, in particular, for determining the concentration of constituents in blood, preferably using a device of the present invention. The method comprises the steps of (a) providing a device comprising a measurement region, a filter and a detection reagent, (b) introducing a fluid into the device, and (c) detecting the concentration of the component using the device. The step of detecting the concentration of the component is preferably carried out using a conventional measuring device, preferably a fluorescence photometer.

The present invention also relates to the use of a device or method of the present invention for determining constituents in blood, such as, bilirubin, free hemoglobin, IL-6 or p24 (HIV protein), CRP. Further preferred is the use for the determination of cell-free DNA. Hemoglobin or bilirubin is preferably measured by measuring the photoabsorption using appropriate wavelengths or by measuring the intrinsic characteristic fluorescence of these substances.

The present invention further relates to a kit comprising at least one device according to the present invention, and a (fluorescence) standard of e.g. B. may contain DNA. The kit preferably further comprises a syringe, preferably with accessories for taking blood from a patient, and / or a measuring device. Furthermore, the kit preferably comprises a manual with interpretation aids. The measuring device may further comprise a device, which is particularly suitable for measuring the samples which were produced with the device. This may include software that allows storage and management of the data. This may include an adapter cable. It may further include a battery pack that allows off-grid measurements.

Figur 1

eine bevorzugte Ausführungsform einer erfindungsgemäßen Vorrichtung mit einem Messbereich wobei Figur 1a eine schematische Draufsicht der Vorrichtung und Figur 1b einen schematischen Schnitt in einer Seitenansicht der bevorzugten Vorrichtung zeigt;

Figur 2

eine schematische Draufsicht auf eine bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung mit zwei parallelen Messbereichen;

Figur 3

eine schematische Draufsicht auf eine weitere bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung mit zwei Messbereichen;

Figur 4

eine schematische Draufsicht auf eine bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung mit drei Messbereichen;

Figur 5

schematische Draufsichten auf weitere bevorzugte Ausführungsformen der vorliegenden Erfindung, wobei Figur 5a eine bevorzugte Ausführungsform mit einem flußoptimierten Messbereich und Figur 5b eine bevorzugte Ausführungsform mit anders flußoptimierten Messbereich darstellen;

Figur 6

eine schematische Draufsicht auf eine bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung mit integriertem Immunassay mit einem Plasma Reservoir;

Figur 7

eine schematische Ansicht einer bevorzugten erfindungsgemäßen Vorrichtung mit einem scheibenförmig ausgeführten Filter, wobei Fig. 7a eine schematische Draufsicht auf die Vorrichtung darstellt und Fig. 7b eine schematische Ansicht der Vorrichtung gemäß Fig. 7a aus Sicht von oben in Fig. 7a; Figur 8 eine schematisch Draufsicht auf eine bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung;

Figur 9

eine schematisch Draufsicht auf eine bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung;

Figur10

eine schematisch Draufsicht auf eine bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung; und

Figur11

eine schematisch Draufsicht auf eine bevorzugte erfindungsgemäße Ausführungsform einer Vorrichtung.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. Hereby show:

FIG. 1

a preferred embodiment of a device according to the invention with a measuring range FIG. 1a a schematic plan view of the device and FIG. 1b shows a schematic section in a side view of the preferred device;

FIG. 2

a schematic plan view of a preferred embodiment of a device according to the invention with two parallel measuring ranges;

FIG. 3

a schematic plan view of another preferred embodiment of the invention of a device with two measuring ranges;

FIG. 4

a schematic plan view of a preferred embodiment according to the invention of a device with three measuring ranges;

FIG. 5

schematic plan views of further preferred embodiments of the present invention, wherein FIG. 5a a preferred embodiment with a flow-optimized measuring range and FIG. 5b represent a preferred embodiment with differently flow-optimized measuring range;

FIG. 6

a schematic plan view of a preferred embodiment of an integrated immunoassay device according to the invention with a plasma reservoir;

FIG. 7

a schematic view of a preferred device according to the invention with a disc-shaped filter, wherein Fig. 7a represents a schematic plan view of the device and Fig. 7b a schematic view of the device according to Fig. 7a from above from above Fig. 7a ; FIG. 8 a schematic plan view of a preferred embodiment of an apparatus according to the invention;

FIG. 9

a schematic plan view of a preferred embodiment of an apparatus according to the invention;

Figur10

a schematic plan view of a preferred embodiment of an apparatus according to the invention; and

Figur11

a schematic plan view of a preferred embodiment of an apparatus according to the invention.

The inventive device, such as in FIG. 1 and 8th is used to detect and preferably determine the concentration of constituents in fluids. Preferably, the fluid is blood and the component to be determined is DNA.

The device after Fig. 1-7 comprises a measuring region 3 and a filter region 5 in fluid communication with the latter. The filter region 5 and the measuring region 3 are preferably connected to one another via a first fluid channel 7. The device 1 preferably further comprises an opening 9, which is preferably designed as a Luer-lock and further preferably with a one-way valve. The opening 9 is preferably located directly at the fluid inlet region of the filter region 5 or is connected thereto via a tube 11, preferably comprising a polymer, preferably of polyvinyl chloride (PVC) or polyethylene (PE).

The filter region 5 is preferably elastic and bag-like and further or additionally preferably made of soft PVC, PE, a mixed or composite polymer. The opening 9 is preferably formed, for example by training as Luer-Lock, that a commercial syringe (not shown) can be connected to it. For example, by a such syringe filled fluid, in particular blood, introduced via the opening 9 and optionally the tube 11 in the filter area 5. In this case, the filter region 5 is preferably designed such that it experiences a predetermined expansion when introducing a certain amount or a certain volume of fluid, which in turn causes a certain pressure on the interior of the filter region 5. Materials to be introduced or required volume and the like are preferably coordinated accordingly. The formation of the opening 9 with a one-way valve prevents the escape of the pressurized, in the fluid input area of the or filter area (s) 5 existing fluid.

As a result of the pressure now prevailing in the filter region 5, the fluid contained in it is forced through a filter, preferably a special membrane, which is furthermore preferably welded into the filter region 5.

In addition to the opening 9, the filter area 5 preferably has an outlet which opens into the fluid channel 7, which in turn leads to the measuring area 3. The filter is preferably arranged in such a way that the pressurized fluid present in the filter region 5 is transported through the filter (not shown) into the fluid channel 7 and thus into the measuring region 3. The fluid thus filtered, preferably the blood plasma, and in particular preferably a predetermined volume of blood plasma, is thus provided in the measuring chamber 3.

The above applies by analogy also to the preferred embodiments Fig. 8-11 , only the parts measuring range 3, fluid channel 7, venting channel 13, as related to the Figures 1-6 described, have been integrated into a part 17, which is preferably completed with a semi-permeable membrane 15.

The apparatus further comprises a detection reagent, preferably Pico-Green ^™ , which is provided in the measuring chamber 3 and / or in the fluid channel 7 in such a way that it collects with the blood plasma or serum flowing through the fluid channel and / or with that collected in the measuring chamber 3 Blood plasma or serum comes into contact and in particular interacts, preferably such that a detection, in particular a determination of the concentration of constituents in the fluid or blood plasma, is made possible.

For this purpose, the device and in particular the volumes of measuring area 3, filter area 5 and fluid channel 7 (respectively part 17) and the properties of the filter area 5 with respect to elasticity and pressure build-up and the filter with respect to the filter and passage properties are coordinated such that in the measuring chamber. 3 or in the fluid channel 7 (or part 17) a predetermined amount of blood plasma is present, which interacts with a predetermined amount of detection reagent.

The measuring chamber 3 and / or the fluid channel 7 (or part 17) is preferably coated on its interior area communicating with the blood plasma, at least partially with a detection reagent, which preferably contains or consists of Pico-Green ^™ or SytoxGreen. Furthermore, a rapidly dissolvable pellet which has a detection reagent can also be positioned in the region of the measuring chamber 3 or the fluid channel 7 (or part 17).

Preferably, a channel, preferably a ventilation channel 13, descends from the measuring area 3. Such a channel preferably opens into a vent opening or vent recess or recess 15, which is preferably arranged in the outer region of the device and is sealed with a semi-permeable membrane (not shown) from the environment. Such a membrane preferably has the property that, seen from the inside of the device, it is permeable to air or gaseous media, but not to liquid media or blood plasma. Accordingly, the channel and the venting area 15 are designed to discharge excess air present outwardly into the environment when filling the filter area 5 or the fluid channel 7 and the measuring area 3 in the device 1, in other words to vent the device 1. This advantageously allows, in particular, the introduction of a defined volume of liquid into the measuring area.

The device 1 according to the invention thus allows automatic filling of the measuring region 3 with a defined amount of blood plasma and mixing with a detection reagent. The device and the fluid present in the measuring range, preferably blood plasma, are accessible in particular to a photo-optical detection. As described above, for example, for the purpose of detecting free DNA by means of an intercalating fluorescent dye such as Pico-Green ^™ .

The detection reagent is preferably applied to the inside of the measuring chamber 5 or the measuring channel, preferably via ink jet, and dried. Alternatively or additionally, the detection reagent is present as dust or pellet present in the measuring chamber or, for example, within the inflow channel as easily soluble material. The device preferably has a diameter of about 10 mm and a length or height of about 50 mm +/- 15 mm.

The measuring area is preferably at least partially transparent or translucent, in particular in order to ensure an optical detection of the component to be detected or determined, preferably by means of a fluorescence measurement.

The foregoing description, in particular with reference to FIG. 1 The same applies accordingly to the preferred embodiments according to FIGS. 2 to 6 (or 7). Here are the preferred embodiments according to FIGS. 2 to 4 Devices that are basically related to the design and operation of FIG. 1 However, these devices have a plurality of measuring regions 3, and preferably 2 or 3 measuring regions, wherein these measuring regions can be arranged differently. Regarding the general structure and the operation is on the above description of FIG. 1 directed. In the case of the embodiment with a plurality of measuring regions 3, it should merely be pointed out that the different measuring regions or the fluid channels 7 emerging from the common filter region 5 and the measuring regions 3 following in the fluid flow direction, Ventilation channels 13 and vent areas 15 are adapted to perform several parallel measurements, or evidence or determinations, each of the measuring ranges 3 and / or fluid channels 7 may have the same or different detection reagents.

The foregoing description, in particular with reference to FIG. 1 The same applies accordingly to the preferred embodiments according to Figures 8-10 , Here are the preferred embodiments according to Figures 8-10 Devices that are basically related to the design and operation of Figure 1-7 correspond to described device,

Preferably, at least one of the measuring ranges, also referred to as a blank value measuring range, is provided for determining a blank value. A blank or zero value is a value used to compare the sample to be measured. For example, blood plasma filtered as a blank can be measured without reagent in the blank measurement range, which then indicates approximately autofluorescence. A calibration value can be formed, preferably with the measurement of a fluid of the same type, which is mixed with standardized amounts of the substance to be measured. More preferably, a calibration value can be formed by measuring a cuvette, which is filled, for example, with a fluid that is optically similar to the blood plasma or else a plastic and has a stable, defined fluorescence. Based on these values, the corresponding measured value of the sample (s) is evaluated. So you can z. B. make a standard daily measurement and make each of the patient a blank and a reading of the analyte of interest.

The corresponding preferred embodiments thus allow the simultaneous performance of parallel, identical measurements or parallel different measurements. As already described in connection with the preferred device of the measuring figure 1, the preferred embodiments also have the same design as in FIG FIGS. 2 to 6 (or 7), the volumes of measuring range 3, filter area 5 and fluid channels 7 and the properties of the Filter range 5 in terms of elasticity and pressure build-up and the filter with respect to the filter and passage properties matched to one another such that in the measuring chambers 3 and in the fluid channels 7, a predetermined amount of blood plasma is present, which interacts with a predetermined amount of Detektionsreagenz (ien).

FIG. 5 again shows a preferred embodiment according to the invention of a device having a measuring chamber, wherein only a modified, flow-optimized measuring region 3 (see FIG FIG. 5a, FIG. 5 ) is shown. This is intended to make it clear that the geometry of the measuring area 3 is not to be understood as limiting, but can experience any desired different configurations as long as the described functionality is ensured.

Also _ FIG. 6 shows a further preferred embodiment of a device according to the invention according to a mixing chamber 25, in which there is preferably a reagent which binds to the / to be measured component (s), a further chamber 20 is arranged downstream. In this chamber 20, a detection reagent, for example an antibody, is firmly bound to a substrate which preferably at least partially contains silicon dioxide (eg glass) or polystyrene or polyurethane. A component to be measured, for example an antigen, then combines in the filter region 5, mixing chamber 25 and / or the channel 7 with a first detection reagent, for example a fluorescence-labeled antibody, and on further flow in the chamber 20 by another to the substrate bound antibody to the substrate and thus determines the presence of the antigen and / or its concentration by fluorescence photometric measurement of the chamber 20. Both detection reagents can bind to different regions of the component to be determined, in the case of antibodies to different epitopes of the antigen.

FIG. 7 shows a schematic view of a preferred device according to the invention with a disc-shaped filter 16. Fig. 7a shows a schematic plan view of the device and Fig. 7b a schematic View of the device according to Fig. 7a from above from above Fig. 7a , The remaining arrangement shown corresponds to those already described.

FIG. 8 shows a schematic plan view of a preferred embodiment of a device according to the invention with a tube-like cuvette 17, which has a z. B. may contain pelleted, readily soluble reagent 23. This device according to the invention also allows automatic filling of the measuring range with a defined amount of blood plasma and mixing with a detection reagent. In this case, advantageously, the combination of the parts already described, measuring range 3, fluid channel 7, venting channel 13 according to the in Figures 1-6 illustrated embodiments integrated into the part 17 and arranged in this. The part 17 is preferably provided with a semi-permeable membrane 15 as a closure. The device and the fluid present in the measuring range, preferably blood plasma, are accessible in particular to a photo-optical detection. The tube-like, translucent measuring cuvette has a length of about 20 mm, an inner diameter of about 1-4 mm and an outer diameter of about 2-6 mm;

FIG. 9 shows a schematic plan view of a preferred embodiment according to the invention of a device in a variant with two tube-like cuvettes 17, in which one can be provided with detection reagent while the other can be measured without reagent, a blank or blank,

FIG. 10 shows a schematic plan view of a preferred embodiment according to the invention of a device in a variant with a mixing chamber 20 which may contain a reagent, wherein the reagent in the chamber 20 particularly advantageously mixes with the filtrate.

FIG. 11 shows a schematic plan view of a preferred embodiment according to the invention of a device in a variant which allows the automatic filling of the measuring range with a defined amount of previously prepared by conventional methods blood plasma. The tube or tube-like The cuvette has a length of approximately 20 mm and an outer diameter of approximately 2-6 mm and may contain a reagent in the region 19.

The present invention thus provides an advantageous apparatus, methods and kit that overcomes the disadvantages of the prior art. In particular, the present invention allows separation of plasma or serum from whole blood and analysis of serum or plasma without the need for centrifugation or similar laboratory processing steps of the recovered serum or plasma. Furthermore, the present invention provides a device, a method and a kit that are easy to handle, safe and reliable, and are particularly suitable for use by laymen or non-medically trained personnel as well as simple and inexpensive to manufacture to perform and / or store.

Claims (35)

Device for detecting, in particular for determining the concentration, of constituents in blood, having a measuring range, a filter and at least one detection reagent for interacting with the constituents. The device of claim 1, wherein the device has an opening for introducing a fluid, wherein the filter is disposed between the opening and the measuring area. Device according to one of claims 1 or 2, wherein the device further comprises a fluid input portion, which is preferably arranged between the opening and the measuring area, more preferably between the opening and the filter. Device according to one of the preceding claims, wherein the ingredient to be detected or determined is a substance occurring in organisms. Device according to one of the preceding claims, wherein the component to be detected or to be determined is a biological molecule selected from the group comprising DNA, RNA, proteins, hormones, cytokines. Device according to one of the preceding claims, wherein the ingredient to be detected or determined is a drug. Device according to one of the preceding claims, wherein the presence and / or concentration of the component in the measuring range by means of luminescence, fluorescence, autofluorescence, chemiluminescence, electrochemiluminescence, spectral absorption photometry and / or bioluminescence can be determined. Device according to one of the preceding claims, wherein the filter is adapted to separate solid components of the blood flowing through the filter, and in particular to separate the solid and liquid phase of the blood from each other. Device according to one of the preceding claims, wherein the fluid input region is formed so that an above ambient pressure is exerted on the blood. Device according to one of the preceding claims, wherein the device is designed such that the blood is introduced under pressure through the filter in the measuring range. Device according to one of the preceding claims, wherein in the device there is a pressure below the ambient pressure. Device according to one of the preceding claims, wherein the detection reagent is provided in the measuring range. The device of any one of the preceding claims, wherein the detection reagent interacts directly or indirectly with the component. Device according to one of the preceding claims, wherein the detection reagent changes its optical properties when interacting with the component to be determined. The device of any one of the preceding claims, wherein the detection reagent is selected from the group comprising Pico-Green ™, Pico-Green ™, Alexa dyes, ethidium bromide and SYBR® or Sytox® dyes. Device according to one of the preceding claims, wherein the opening comprises a one-way valve. Device according to one of the preceding claims, wherein the opening has a Luer lock. Device according to one of the preceding claims, wherein the fluid input region is formed or delimited by a, preferably elastic, film. Device according to one of the preceding claims, wherein the device is a one-way device. Apparatus according to any one of the preceding claims, wherein the filter is adapted to separate serum or plasma from the blood. Device according to one of the preceding claims, wherein the device is compatible with commercially available detection devices. Device according to one of the preceding claims, wherein the device has the dimensions of a commercially available cuvette or other commercially available measuring vessel and preferably has a diameter of about 10mm and / or a length of about 50mm +/- 15 mm or by means of an adapter to the size of a commercial cuvette is adjusted. Device according to one of the preceding claims, wherein the device comprises at least one venting device. Device according to one of the preceding claims, wherein the venting device is connected to the measuring chamber through a narrow groove or a gap. Device according to one of the preceding claims, wherein the measuring range of the device is in the form of a tube, preferably with a venting device. Device according to one of the preceding claims, wherein the device has at least one second measuring range. Device according to one of the preceding claims, wherein the device has at least one area for providing a blank or calibration value. Device according to one of the preceding claims, wherein the component to be determined interacts with two detection reagents. The device of claim 28, wherein the first detection reagent in the device is in front of the measurement area. Apparatus according to claim 28 or 29, wherein the second detection reagent is immobilized to a certain area in the measuring area. Method for detecting, in particular for determining the concentration, of constituents in blood, preferably using a device according to one of the preceding claims, comprising the steps of providing a device with a measuring range and a detection reagent, introducing blood into the device, and detecting the resp Measuring the concentration of the ingredient using the device. The method of claim 31, wherein the blood is introduced by means of a syringe into the device, wherein preferably the pressure required for filtering is applied. Use of a device or a method according to any one of the preceding claims for the determination of constituents in blood. A kit comprising the device of any one of claims 1 to 30 and a fluorescence standard. Device according to one of the preceding claims, wherein the component to be detected or to be determined is a protein.

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