Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/02—Adapting objects or devices to another
  • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
  • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/04—Closures and closing means
  • B01L2300/046—Function or devices integrated in the closure
  • B01L2300/049—Valves integrated in closure
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0681—Filter
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
  • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum

Definitions

• 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.
• US 5,186,843 describes a material for separating plasma or serum from blood.
• 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.
• US 4,816,224 a device for the separation of plasma or serum from whole blood is described, which may be designed differently. Glass fibers, which occupy a large volume, serve for separation.
• the device may comprise a plurality of filter layers.
• a device 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 which detects the components in the
• the device is primarily intended for obtaining blood plasma or serum for further study.
• devices as described above are primarily used to provide small amounts of plasma or serum for further analysis.
• Measurement results are often designed only indicative values can be obtained and not infrequently only statements about the presence or absence of measured components of a liquid can be made. For example, test strips only provide information on whether or not a substance is present within the detection capacity.
• 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.
• 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.
• the present invention preferably relates to a device for detecting, in particular for determining the concentration, of constituents in blood, having a measuring region, a filter and / or filter region, at least one detection reagent for interacting with the constituents, an opening for introducing a fluid, wherein the filter is disposed between the opening and the measuring area, and a fluid input area, wherein the fluid input area and / or the filter region is at least partially formed or delimited by a preferably elastic region, preferably comprising a film.
• the fluid input region is preferably arranged between the opening and the filter.
• Detection of, for example, substances means, in particular, the determination of the presence or absence of a substance, whereby the detection limit of the measurement method determines the result within the scope of the desired accuracy.
• the device of the present invention can also be used as components of others Fluids are herein to be understood as liquids having solid constituents or suspensions,
• the fluid is a bodily fluid
• Body fluids are, for example, blood, cerebrospinal fluid, urine, serous fluids, saliva, semen, or diseased chair. in particular from lines, streams or lakes etc.
• 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.
• the measuring range is preferably downstream of the filter and is in fluid communication therewith. It may be directly adjacent to the filter or spaced therefrom.
• the area directly adjacent to the filter that receives the filtrate is also referred to as the filtrate area.
• the filtrate area can either completely or partially correspond to the measuring area or to the filter (s) downstream of the elements which receive the filtrate and which are arranged in front of the measuring area.
• 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 comprise or consist of any material suitable for separating blood or other solid-component fluids.
• the filter may include, for example, polyethylene terephthalate (sold, for example, by Sekisui in a serum recovery product) or polysulfone (eg, Vivid Plasma Separation Membrane (formerly BTSSP) sold by PaII) or based on these materials be.
• 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 the When filtering blood, the filter material should not cause hemolysis or bind analytes.
• detection reagent is used in particular to detect or detect a substance with which the presence and / or concentration of another substance, preferably a substance present in a fluid, eg blood, plasma or water, can be detected or detected
• a detection reagent preferably has the property of allowing or causing a detectable reaction under certain conditions
• a detection reagent preferably interacts directly with the substance to be determined, whereby either a covalent or a non-covalent bond with this substance
• the detection reagent preferably fluoresces to a greater extent only through the binding to the analyte.
• the device has an opening for introducing a fluid, wherein the filter is arranged between the opening and the measuring area. Furthermore, the device has a fluid input region.
• the fluid input area is preferably arranged between the opening and the measuring area, more preferably between the opening and the filter.
• the filter region further comprises the filter, parts or the entirety of the fluid input region, the filtrate region and / or the measuring region.
• the filter area preferably has a capacity of between about 200 and about 2000 ⁇ l. Depending on the fluid or, preferably, the amount and nature of the blood, up to 200 ⁇ l 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 ⁇ l and about 80 ⁇ l, preferably between about 20 ⁇ l and about 60 ⁇ l and most preferably about 40 ⁇ l serum or plasma.
• the fluid input region and / or filter region is preferably formed or delimited at least partially or completely by a, preferably elastic, film.
• the film is preferably made of an artificial or natural polymer or mixed or copolymer.
• Examples of such polymers and copolymers or copolymers are PVC, ethylene-vinyl acetate copolymer film, polyethylene, polyethylene copolymer, polypropylene, polypropylene copolymer, a mixture of these polymers or block polymers, coextrusions, multilayer films and also films with no smoother Surface.
• the film is preferably the boundary of the filter area.
• the filter is preferably connected in the filter area with the film limiting the film area, eg welded or glued, that the downstream of the filter region that receives the filtrate is completely separated by the filter from the fluid input area , Before filling the device according to the invention, the filter in space-saving form, for. B. folded, or even flat against the filter area limiting film.
• the fluid input region and the region downstream of the filter have only a slight and preferably no internal volume prior to filling. If the fluid input region is filled with sample, the pressure at which the sample is introduced and the elasticity of the film results in the inflation of the fluid input region surrounded by the film. This pressure is held in the fluid input area so that the sample is pushed through the filter. By filtration, the downstream of the filter area is filled with filtrate. If this area is also covered by the elastic film, the result of this preferred embodiment of the invention is a particularly small dead space volume on the filtrate side, thereby making it possible to particularly advantageously use very small amounts of filtrate. Furthermore, the fragile filter membrane preferably adheres to the inside of the foil delimiting the sachet and thus experiences advantageous mechanical support.
• 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 is particularly advantageous in that it provides a device that is small, that can be read out with commercially available devices, and / or the separation of the solid components from fluids, preferably from blood, with the simultaneous measurement of in the liquid phase links existing components.
• 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.
• Pulsma 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.
• each substance can be determined using suitable detection reagents.
• the Component 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.
• nucleic acids comprising DNA and RNA in their single and / or double-stranded forms.
• the component to be detected or to be determined is a biological molecule selected from the group comprising DNA, RNA, proteins,
• Hormones may be free or associated with other proteins.
• DNA in plasma or serum may also occur as a complex with histones and / or elastase as well as microsatellites.
• 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.
• particles are retained by the filtration, while those contained in microorganisms
• Nucleic acid can be made accessible to a measurement by suitable fluorescent dyes.
• the ingredient to be detected or determined is a drug.
• Drugs are substances that can be administered to an individual combat a pathological condition or disease. Drugs may also be the abovementioned biological molecules.
• 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.
• 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 ⁇ l range (eg measurement by NanoDrop in the range from 2 ⁇ l). The lower measurement limit for DNA is z. At about 10 ng / ml for the fluorescence method.
• 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.
• 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, the apoptosis of cells, among other things, of blood cells is induced. The therapeutic success can thus be detected directly and finely graded with the present invention.
• 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.
• the filter is adapted to separate solid components of the fluid flowing through the filter, and in particular the solid and liquid phases to separate the fluid from each other.
• Some filter materials can be very brittle or fragile, so stabilization is necessary.
• 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.
• 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.
• 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.
• a pressure which is below the ambient pressure prevails in the device, preferably one
• the low pressure or the vacuum causes the sample to be conducted or sucked into the device without externally applied force, and inside the device
• the measuring range is preferably made of a preferably elastic material, which endeavors to assume a defined position or shape.
• the measuring range is preferably made of a preferably elastic material, which endeavors to assume a defined position or shape.
• the measuring range is preferably made of a preferably elastic material, which endeavors to assume a defined position or shape.
• Inner volume of the measuring chamber is smaller than that of the defined position, so that it is e.g. unfolded during the introduction of the sample or the sample is sucked in during deployment.
• the measuring range is made of an inelastic
• 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.
• the detection reagent is provided in the filter or in spatial proximity to the filter.
• 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.
• 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.
• the detection reagent is selected from the group also including cyanine dyes (Pico-Green TM ([2- [N-bis (3-dimethylaminopropyl) amino] -4- [2,3-dihydro-3-methyl - (benzo-1,3-thiazol-2-yl) methylidene] -1-phenyl-quinoline]; see also Zipper et al., 2004, Nucleic Acids Research 32 (12)), TOTO® (e.g.
• Alexa® - eg, 2,3,5,6-tetrafluorophenyl ester (Alexa Fluor® 488 5-TFP; a review of Alexa dye is from Berlier et al.
• a quantitative determination of a substance in the measuring range is carried out.
• the detection reagent when interacting with the target molecule, it is advantageous for the detection reagent, when interacting with the target molecule, to change its properties such that it becomes detectable.
• 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.
• fluorophores can be used within the scope of the invention.
• 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
• Pico-Green TM and / or SYTOX Green are preferred.
• the dose of Pico-Green TM is preferably about 0.01 to 5 ⁇ l of reagent, preferably 0.05 to 2 ⁇ l, more preferably 0.1 to 0.5 ⁇ l of reagent, most preferably 0.15 to 0.3 ⁇ l Reagent per measurement at approx. 40 ⁇ l sample volume.
• the amount of reagent used is adjusted to the sample volume.
• the amount of solid in its content of detection reagent corresponds to the content of dissolved solid in o.g. Volumes.
• the opening of the device has a one-way valve.
• the opening has a Luer lock.
• the device is a disposable device.
• 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.
• Such substances may be, for example, titanium dioxides or colestyramine.
• the device in particular with respect to their dimensions, compatible with commercially available detection devices.
• detection devices include, in particular, commercially available photometers, such as, for example, the Picofluor, TBS380 (Turner Biosystems, USA) or the qubit (Invitrogen, USA).
• 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.
• the expression "commercially available cuvette” preferably corresponds to a cuvette with a diameter of about 10 mm and / or a length of about 50 mm +/- 15 mm.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• PCR PCR
• 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.
• the component to be determined interacts with two detection reagents.
• 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.
• 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.
• 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 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 immobilized in a certain area of the measurement area. By accumulating the complex at this point, the constituent to be determined becomes detectable.
• 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.
• 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.
• the detection reagents are preferably antibodies or antibody fragments.
• the antibody is coupled to a substance to be detected.
• a substance to be detected for example, R-phycoerythrin (PE), fluorescein isothiocyanate (FITC), PE-Cy5 allophycocyanin (APC), PE-Texas Red TM, Peridinin Chlorophyll Protein (PerCP), PerCP-Cy5.5, APC-Cy7, and / or Texas Red TM, 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.
• endogenous substances such as interleukin 6 (IL-6), hemoglobin, bilirubin, CRP, lactoferrin, procalcitonin, AT-III, protein C, p24 (HIV) or antibodies.
• 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 components 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 withdrawing blood from one Patient, and / or a measuring device.
• 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.
• Figure 1 shows a preferred embodiment of a device according to the invention with a measuring range wherein Figure 1a shows a schematic plan view of the device and Figure 1 b shows a schematic section in a side view of the preferred device;
• Figure 2 is a schematic plan view of a preferred embodiment of an apparatus according to the invention with two parallel measuring areas;
• Figure 3 is a schematic plan view of another preferred embodiment of the invention of a device with two measuring ranges;
• Figure 4 is a schematic plan view of a preferred embodiment of the invention of a device with three measuring ranges;
• Figure 5 is a schematic plan view of further preferred embodiments of the present invention, wherein Figure 5a represents a preferred embodiment with a flow-optimized measuring range and Figure 5b is a preferred embodiment with different flow-optimized measuring range;
• FIG. 6 shows a schematic plan view of a preferred embodiment of an apparatus according to the invention with an integrated immunoassay with a plasma reservoir;
• FIG. 7 shows a schematic view of the device according to FIG. 7a but filled with a fluid and therefore with an elastically curved fluid input area.
• FIG. 7a shows a schematic plan view of the device;
• FIG. 7c shows FIG. 7a as viewed from above.
• Figure 8 is a schematic plan view of a preferred embodiment of a device according to the invention.
• Figure 9 is a schematic plan view of a preferred embodiment of a device according to the invention.
• FIG. 12a shows a schematic side view of a preferred device according to the invention, wherein the filter 4 separates the fluid input region 5 and the subsequent filtrate region;
• FIG. 12b shows a schematic side view with the construction of the device described in FIG. 12a after filling with a sample to be filtered, similar to the device according to FIG. 7b;
• Figure 12c is a schematic plan view of the structure of the device described in Figure 12a, b.
• the device according to the invention serves to detect and preferably to determine the concentration of constituents in fluids.
• the fluid is blood and the component to be determined is DNA.
• the device according to FIGS. 1-7 comprises a measuring region 3 as well as 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).
• PVC polyvinyl chloride
• PE polyethylene
• 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.
• fluid filled in by such a syringe, in particular blood is introduced into the filter region 5 via the opening 9 and optionally the tube 11.
• 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.
• the fluid contained in it is forced through a filter, preferably a special membrane, which is furthermore preferably welded into the filter region 5.
• 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 apparatus further comprises a detection reagent, preferably Pico-Green ⁇ TM, which is provided in the measuring chamber 3 and / or in the fluid channel 7 such that it communicates with the blood plasma or serum flowing through the fluid channel and / or with that in the measuring chamber 3 collected blood plasma or serum comes into contact and in particular interacts, preferably in such a way that a detection, in particular a determination of the concentration of components in the fluid or blood plasma, is made possible.
• a detection reagent preferably Pico-Green ⁇ TM
• 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.
• 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).
• a channel preferably a ventilation channel 13 descends from the measuring area 3.
• a channel preferably opens in a vent or
• Vent recess or recess 15 preferably in the outdoor area of
• a semi-permeable membrane (not shown) from the environment.
• a semi-permeable membrane preferably has the property that it is permeable to air or gaseous media seen from the inside of the device, but not for liquid
• Venting area 15 designed to dissipate excess air to the outside in the environment when filling the filter area 5 and 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
• the device 1 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.
• a photo-optical detection for example, according to the above description, for the purpose of detecting free DNA by means of an intercalating fluorescent dye such as _. _ TM, for example, Pico-Green.
• the detection reagent is preferably on the inside of the measuring range 3, z. B. a measuring chamber, or the measuring channel, preferably via inkjet or equivalent to the inkjet printer technology, 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.
• FIGS. 2 to 4 show devices which fundamentally correspond in terms of structure and functionality to the device described in connection with FIG. 1, but which have a plurality of measuring regions 3, and preferably 2 or 3 measuring regions, whereby these measuring regions can be arranged differently , With regard to the general structure and the operation is made to the above description of Figure 1.
• each of the measuring regions 3 and / or fluid channels 7 can have the same or different detection reagents.
• the preferred embodiments according to FIGS. 8-10 show devices which correspond in principle to the construction and mode of operation of the device described in connection with FIGS. 1-7.
• at least one of the measuring ranges also referred to as 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.
• the volumes of the measuring area 3, filter area 5 and fluid channels 7 as well as the properties of the filter area 5 with regard to elasticity are also shown in the preferred embodiments as shown in FIGS. 2 to 6 (or 7) 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 with a measuring chamber, wherein only a modified, flow-optimized measuring region 3 (see FIG. 5a, FIG. 5) is shown by way of example. This is to clarify that the geometry of the measuring range 3 is not as is to be understood restricting, but can learn any different configurations as long as the functionality described is preserved.
• FIG. 6 also shows a further preferred embodiment of a device according to the invention in accordance with a mixing chamber 25, in which there is preferably a reagent which binds to the component (s) to be measured, a further chamber 20 is arranged downstream.
• a detection reagent e.g. an antibody bound tightly to a substrate which preferably at least partially contains silica (e.g., glass) or polystyrene or polyurethane.
• a component to be measured e.g. an antigen, then combines in the filter section 5, mixing chamber 25 and / or channel 7 with a first detection reagent, e.g.
• a fluorescently labeled antibody is further fixed in the chamber 20 by another antibody bound to the substrate to the substrate and thus determines the presence of the antigen and / or its concentration by a 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 plan view of the device
• FIG. 7b shows a schematic view of the device according to FIG. 7a but filled with a fluid and therefore with elastic domed fluid input area
• FIG. 7c shows a schematic view of the device according to FIG. 7a as viewed from above in FIG. 7a. The remaining arrangement shown corresponds to those already described.
• Figure 8 shows a schematic plan view of a preferred embodiment according to the invention of a device with a tube-like cuvette 17, which has a z. B. may contain pelleted, readily soluble reagent 23.
• This inventive device allows the automatic filling of the measuring range with a defined amount of blood plasma and mixing with a Detection reagent.
• the combination of the already described parts measuring range 3, fluid channel 7, venting channel 13 is integrated according to the embodiments shown in Figures 1-6 in 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 of a device according to the invention 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 value,
• 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, the reagent in the chamber 20 particularly advantageously mixing with the filtrate.
• FIG. 11 shows a schematic plan view of a preferred embodiment of a device according to the invention in a variant which allows the automatic filling of the measuring area with a defined amount of blood plasma prepared beforehand by conventional methods.
• the tube-like or cuvette-like cuvette has a length of about 20 mm and an outer diameter of about 2-6 mm and may contain a reagent in the region 19.
• FIG. 12a shows a schematic side view of a preferred device according to the invention, wherein the filter 4 separates the fluid input region 5 and the filtrate region, which terminates in a part 7 or 17.
• the filter is preferably with the fluid input region as well as the measuring range and / or the walls or films bounding the filtrate region are connected without gaps, preferably by gluing or welding.
• the part 7 or 17, comprises z. B. a capillary for venting and / or measurement and / or the measuring range 3,
• FIG. 12b shows a schematic view after the filling of the device according to FIG. 12a.
• FIG. 12c shows the broad-side view of FIGS. 12a, b.
• the devices have elastic walls, preferably formed from films or having one or more film areas, which limit the fluid input area 5 and the filtrate area.
• the filter 4 can both closely conform to the walls of the device and be folded. In both states, the potentially fragile filter membrane experiences mechanical support.
• the device further comprises, for example, a capillary or orifice 17 suitable for transporting, measuring and / or removing the filtrate.
• the device is preferably filled via the opening 9 or the fluid input area.
• a syringe is preferably used.
• the opening 9 has a one-way valve, for example a Luer lock.
• the sample material pressurized by the stretched film or stretched elastic material is thus forced through the filter and filtered.
• the filtrate exiting on the other side of the filter thus passes through the filter into the filtrate area and / or measuring area.
• This region is also preferably at least partially formed by an elastic material, for example an elastic film, so that its volume is formed as a function of the incoming filtrate.
• the present invention thus provides an advantageous apparatus, methods and kit that overcomes the disadvantages of the prior art.
• 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.
• 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.

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• Clinical Laboratory Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
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• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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• 2007
  • 2007-10-31 EP EP07021342A patent/EP2055384A1/de not_active Withdrawn
• 2008
  • 2008-10-31 WO PCT/EP2008/009219 patent/WO2009056340A2/de not_active Ceased
  • 2008-10-31 US US12/740,817 patent/US20100261223A1/en not_active Abandoned
  • 2008-10-31 CN CN2008801144648A patent/CN101883634A/zh active Pending
  • 2008-10-31 JP JP2010531462A patent/JP2011501201A/ja active Pending
  • 2008-10-31 EP EP08845028A patent/EP2205355A2/de not_active Withdrawn

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