Rapid Test for the Detecting Pathogens and Cells and Method

Abstract

The invention relates to a test system comprising at least one of the following means: permeabilising means (110) and/or lysis of at least one pathogen (10) and/or at least one cell (10), means for capturing (80) and/or making (90) parts (20 and/or 40 and/or 50) of the pathogens (10) and/or cells (10), means for localising, immobilising and/or enriching (120) at least one component (40 and/or 50 and/or 20 and/or 10) of a pathogen (10) and/or a cell (10), means for image processing (160) preferably comprising an optical magnifying unit (161), enabling an optical reading out of at least one means for localising, immobilising and/or enriching (120) can be carried out.

Claims

1-15. (canceled)

16. A test system, the test system comprising at least one of the following means: means for at least permeabilizing or lysing at least one pathogen or at least one cell; means for at least capturing or labeling parts at least of the pathogens or cells; means for at least localizing, immobilizing or enriching at least one component at least of a pathogen or of a cell; means for image processing, whereby an optical read-out of at least one means for at least localization, immobilization or enrichment can be carried out.

17. The test system according to claim 16, wherein the means at least for localization, immobilization or enrichment comprises elements for receiving a sample fluid.

18. The test system according to claim 16, wherein the means for permeabilization can be used to achieve that ligands have at least access to at least one target structure, such as a biomolecule, or to at least one structure of a pathogen or to at least one cell/pathogen.

19. The test system according to claim 16, wherein at least the means at least for localizing, immobilizing or enriching can at least be used for at least one component of pathogens or cells, wherein at least one component of at least pathogens or cells can be enriched or the means at least for localization, immobilization or enrichment comprises a microfluidic structure at least for dissolving or adding or mixing or incubating substances.

20. The test system according to claim 16, wherein the means at least for localization, immobilization or enrichment comprises at least one microfluidic structure for incubating a sample fluid with at least a capture ligand or a labeling ligand, and at least one detection area, wherein at least one component of at least one labeled pathogen or of at least one labeled cell can be received on the means at least for localization, immobilization or enrichment.

21. The test system according to claim 16, wherein the means at least for localization, immobilization or enrichment comprises at least one microfluidic structure made of PET into which aqueous liquid, can flow, having no further coating.

22. The test system according to claim 16, wherein the means for binding at least comprises at least one capture ligand or at least one labeling ligand for labeling.

23. A test method for diagnosing diseases, the method comprising at least one of the following steps: a) applying a sample fluid, in particular a liquid, to the means at least for localization, immobilization or enrichment; b) at least applying or releasing a means for permeabilization onto the means at least for localization, immobilization or enrichment, whereby it is ensured that ligands have access to structures at least of at least one pathogen or of at least one cell contained in the sample fluid; c) at least applying or releasing at least a labeling ligand to or on at least one antibody, or a DNA stain, or at least applying or releasing a capture ligand to or on the means at least for localization, immobilization or enrichment; d) labeling at least one component at least of at least one pathogen or at least one cell; e) enriching at least one component at least of a pathogen or of a cell; and f) detecting at least the pathogen or the cell at least through microscopic magnification or image recording.

24. The test method according to claim 23, wherein a detection at least of infectious diseases, diseases caused at least by fungi, viruses or bacteria, or pathogens or agents can be achieved.

25. The test method according to claim 23, wherein the means at least for localization, immobilization or enrichment can be used to detect malaria pathogens in a blood sample.

26. The test method according to claim 23, wherein the means at least for localization, immobilization or enrichment can be used to detect malaria pathogens, in a blood sample

27. The test method according to claim 23, wherein at least a sample can be mixed with a buffer, or solid components can be separated, for example at least by way of filtration, sedimentation or centrifugation, or pathogens, such as legionella, or eggs of the pathogens, such as Ascaris, Trichiuris, Amylostoma, Taenia, can be permeabilized at least using a means for lysis or permeabilization, or at least pathogens, eggs of the pathogens or the components thereof can be enriched by way of a means at least for localization, immobilization or enrichment, such as a filter structure or magnetic particles, or these can be labeled with a labeling ligand, or can be detected by way of imaging microscopy.

28. The test method according to claim 23, wherein at least the means at least for localization, immobilization or enrichment comprises at least one microcuvette, in which a sample liquid, preferably a defined amount of whole blood from the finger pad, can be received, or the sample liquid can preferably be mixed with lyophilized substances, such as at least an anticoagulant, or a capture ligand, with or without magnetic beads or one or more means at least for labeling or stabilizers or a means at least for lysis or permeabilization, wherein the mixing can be carried out, for example, by applying a switchable external magnetic field, so that the magnetic particles contained therein can be conducted through the solution, wherein, preferably subsequent immobilization of the pathogens bound to the means for immobilization or microscopic detection through a vision panel are carried out.

29. The test method according to claim 23, wherein at least the means at least for localization, immobilization or enrichment comprises at least one microfluidic structure in combination with a filter structure, such as preferably an absorbent structure, or a liquid can be added to the microfluidic structure, or the sample liquid can preferably be mixed with lyophilized substances, such as at least an anticoagulant or one or more means for labeling, such as antibodies including a fluorescent tag or gold particles, or stabilizers, or a means for lysis and/or permeabilization, wherein, preferably the liquid can be brought in contact with the filter structure, for example at least by opening a ventilation opening or, for example, at least by displacing or by bending a chip or by overcoming a flow barrier, wherein, preferably the liquid from the capillary can thereafter be absorbed by the filter structure, wherein, preferably a capture ligand can be immobilized on the filter structure, wherein, preferably the pathogen can be brought in contact with the capture ligand, wherein, preferably a band is created, which is composed at least of labeled pathogen or the components thereof, and wherein, preferably this band can at least be analyzed microscopically or photographically or visually.

30. Use of at least a test system comprising at least one of the following means: means for at least permeabilizing or lysing at least one pathogen or at least one cell; means for at least capturing or labeling parts at least of the pathogens or cells; means for at least localizing, immobilizing or enriching at least one component at least of a pathogen or of a cell; means for image processing, whereby an optical read-out of at least one means for at least localization, immobilization or enrichment can be carried out or test method for diagnosing diseases, comprising at least one of the following steps: a) applying a sample fluid, in particular a liquid, to the means at least for localization, immobilization or enrichment; b) at least applying or releasing a means for permeabilization onto the means at least for localization, immobilization or enrichment, whereby it is ensured that ligands have access to structures at least of at least one pathogen or of at least one cell contained in the sample fluid; c) at least applying or releasing at least a labeling ligand to or on at least one antibody, or a DNA stain, or at least applying or releasing a capture ligand to or on the means at least for localization, immobilization or enrichment; d) labeling at least one component at least of at least one pathogen or at least one cell; e) enriching at least one component at least of a pathogen or of a cell; and f) detecting at least the pathogen or the cell at least through microscopic magnification or image recording for diagnosing diseases.

31. The test system according to claim 21, wherein the means can be closed at least partially by a film and/or divided into one or more areas, which initially is closed, whereby the liquid does not continue to flow further through the same and through which the liquid can flow after piercing a ventilation opening behind it.

32. The test system according to claim 31, wherein at least a subdivision into a sample receiving area, in which a defined sample volume, such as whole blood from the finger pad, can be received, or a mixing area in which the sample can preferably be mixed with lyophilized substances, such as an anticoagulant, and/or a capture ligand, with or without magnetic beads, and/or one or more labeling ligands and/or stabilizers and/or a means for lysis and/or permeabilization, or a detection area, in which the pathogens can at least be localized or enriched or immobilized.

33. The test system according to claim 22, wherein this at least includes at least one antibody, an aptamer or a ligand or a microparticle, nanoparticle, magnetic microparticle or magnetic nanoparticle, which can be coupled to an antibody, an aptamer or a ligand, wherein at least preferably at least one magnetic nanoparticle or a metal core can be bound by way of a coupled capture ligand to at least one component at least of pathogens or cells.

34. The test method according to claim 25, wherein at least one capture ligand which can preferably be at least coupled to at least one magnetic microparticle or nanoparticle or at least one labeling ligand, preferably at least one fluorescent labeled antibody at least against plasmodial proteins or at least one DNA stain against plasmodial DNA is able to access to at least one structure in infected erythrocytes after allowing a means for permeabilization, to act, so as to detect, at least one structure at least of plasmodia or of erythrocytes infected with at least one or more plasmodia, or to at least capture at least one infected erythrocyte or at least one structure of plasmodia at least by way of retention or magnetic separation in a means at least for localization, immobilization or enrichment.

35. The test method according to claim 26, wherein at least after allowing a means for permeabilization, access is made possible to pathogens having an enveloping parasitophorous vacuole in infected erythrocytes, and preferably these can be released from the erythrocytes, and at least one structure for at least one capture ligand, preferably at least one antibody against plasmodial proteins such as RAP1 or EXP2, such as the monoclonal antibody anti-RAP-1, Clone 2.29, or the monoclonal antibody anti-EXP-2, Clone 2.2, which can preferably be coupled to at least one magnetic microparticle or nanoparticle, in particular a Dynabead or Dynabead Myone, or by at least one or more labeling ligands, preferably at least one or more labeled antibodies, such as the monoclonal antibody anti-RAP-1, Clone 2.29, or the monoclonal antibody anti-EXP-2, Clone 2.2, against plasmodial proteins such as RAP1 or EXP2, or at least one, preferably fluorescent, DNA stain against plasmodial DNA, so as to detect, preferably, at least one structure at least of plasmodia or of erythrocytes infected with at least one or more plasmodia, or at least to immobilize at least one infected erythrocyte or at least one structure of plasmodia at least by way of retention or magnetic separation in a means at least for localization, immobilization or enrichment, such as a microtiter plate having at least 96, and preferably 1536 wells, a microcuvette or a simple fluidic structure.

Description

[0035] Further measures improving the invention will be apparent from the following description of several exemplary embodiments of the invention, which are schematically illustrated in the figures. All of the features and/or advantages that are apparent from the claims, the description and/or the drawings, including design details, arrangements in terms of space, and method steps, can be essential to the invention, both alone and in a wide variety of combinations. It should be noted that the figures are only of a descriptive nature and not intended to limit the invention in any way. In the drawings:

[0036] FIG. 1a shows a schematic representation of a target cell prior to permeabilization;

[0037] FIG. 1b shows a schematic representation of a target cell after permeabilization;

[0038] FIG. 2a shows a schematic representation of a reaction vessel as a means for localization, immobilization and/or enrichment;

[0039] FIG. 2b shows a schematic representation of a microchannel as a means for localization, immobilization and/or enrichment;

[0040] FIG. 2c shows a schematic representation of a microchannel having a retention device;

[0041] FIG. 3 shows a schematic representation of a recorded image and processing after the permeabilization, labeling and capture of pathogens and/or cells; and

[0042] FIG. 4 shows a schematic representation of a test system.

[0043] FIG. 1a shows a pathogen and/or an infected cell 10 including specific inner target structures 40, 50, such as an antigen, a protein, a lipid, a glycosaccharide chain, a nucleic acid chain, a peptide or other biomolecules. This may be a pathogen 10 such as a bacterium, which has a compartmentalizing structure 30, for example a cell membrane and/or a cell wall, which essentially partitions the inside of the cell from the outside of the cell. It includes target structures 40, 50 specific to this bacterial species, for example on one or more inner structures 20, such as a thylakoid, ribosome, plasmid, chlorosome, a basal apparatus of a flagellum, a nucleotide, a cytoplasmic side of the cell membrane and/or other components on which target structures, such as antigens, can be found. A capture ligand 60 added to the outer solution and/or dissolved therein, such as a specific antibody, for example including a coupled magnetic nanoparticle 70, such as a Turbobead, and/or a labeling ligand 80, such as a specific antibody having a coupled tag 90, such as a fluorophore, a quantum dot, an enzyme or a gold particle and/or other tag, cannot penetrate into the interior of the bacterium due to the compartmentalizing structure 30, such as a cell wall and/or a lipid membrane and/or another structure.

[0044] The cell 10 can likewise be an erythrocyte 10 infected with a malaria pathogen 20 (Plasmodium spec.), for example. The target structures 40, 50 can likewise be Plasmodium-specific structures, which are arranged inside the erythrocyte.

[0045] The erythrocyte is enveloped by a cell membrane, which is a compartmentalizing structure 30 and essentially separates the inside of the cell from the outside of the cell. It includes target structures 40, 50 specific to this bacterial species, for example on one or more inner structures 20, such as plasmodial proteins on the cytoskeleton and/or on an inner membrane. A capture ligand 60 added to the outer solution and/or dissolved therein, such as a specific antibody, for example including a coupled magnetic nanoparticle 70, such as a Turbobead, and/or a labeling ligand 80, such as a specific antibody including a coupled tag, such as a fluorophore, a quantum dot, an enzyme or a gold particle, cannot penetrate into the interior of the erythrocyte due to the compartmentalizing structure 30.

[0046] FIG. 1b shows a cell 10 after a means for permeabilization 110, such as saponin, pore inducers, lysing agents, import-triggering agents, hypotonic or hypertonic solutions and/or other substances, has been added. Due to the means for permeabilization, openings 31 in the form of cracks, pores, holes, destabilized membrane areas, for example without cholesterol, and/or a receiving process 31, such as transport mechanisms, can develop in the compartmentalizing structure 30. As a result, the capture ligand(s) 60, including a coupled magnetic nanoparticle 70, and/or the labeling ligand 80, including a coupled tag 90, added to the outer solution can penetrate the cell and/or the pathogen and bind therein specifically to the respective target structure 40 and/or 50. The capture ligand 60 and the labeling ligand 80 preferably have no cross reactivity, which is to say they bind to different target structures 40, 50, whereby preferably a sandwich structure can be created. The binding of the labeling ligand 80, such as a fluorescent labeled antibody, to a target structure 40, such as an antigen typical of a pathogen, can preferably result in immunostaining.

[0047] FIG. 2a shows a schematic representation of a reaction vessel 121 as a means for localization, immobilization and/or enrichment containing a sample including cells 10, 11. The cells 10 can preferably represent target cells, and the cells 11 can represent non-target cells. The reaction vessel 121 can be micro reaction vessel, for example, such as an Eppendorf reaction vessel, and/or a chromatography column. An associated magnet 130, such as a high gradient magnet and/or a permanent magnet and/or a solenoid and/or another magnet, can generate a magnetic attractive force 131 in the reaction vessel 121. This attractive force 131 can act on the magnetic nanoparticles 70, whereby these and the target structure 50 bound by way of the capture ligand are attracted and can be enriched and immobilized in spatial proximity to the magnet, to the extent this is possible in the reaction vessel 10. The bound target structure 50 can preferably be joined to components and/or the majority of the cell and/or of the pathogen 10, so that the bound target structure 50 is enriched with attached further components in spatial proximity to the magnet, such as on the wall of the reaction vessel facing the magnet. Non-target cells, such as non-infected erythrocytes, cannot be bound by capture ligands and cannot be enriched. Structures of the target cell can be labeled by way of binding of a labeling ligand, such as a fluorescent labeled antibody and/or a fluorescent labeled DNA stain.

[0048] FIG. 2b shows a schematic representation of a microfluidic structure 140 as a means for localization, immobilization and/or enrichment, containing a sample therein, using a capture ligand including a magnetic nanoparticle. A microfluidic structure is composed of a part containing capillaries and/or microstructures and/or one or more microchannels. The sample liquid containing cells 10, 11 therein can flow through the microfluidic structure 140 in the direction of flow 141. An associated magnet 130 can generate a magnetic force 131 in an area of the microfluidic structure. This force can attract the magnetic nanoparticles 70, as shown in FIG. 2a, and thereby act on the coupled capture ligand 60 and a bound target structure 50 and/or preferably a bound majority of the structure 20 and/or 10. This can result in an enrichment of the target structure, and preferably of the cells 10 and/or the components of the microfluidic structure 140, in particular at the top, the side walls and/or the bottom. Cells 11, such as non-infected erythrocytes, cannot be bound by capture ligands 60 and cannot be enriched. A target structure or target structures 40 of the target cell 10 can be labeled by way of binding of a labeling ligand 80, such as a fluorescent labeled antibody and/or a fluorescent labeled DNA stain.

[0049] FIG. 2c shows a schematic representation of a microfluidic structure 140 as a means for localization, immobilization and/or enrichment, containing a sample therein, and a retention device in the microfluidic channel. The sample liquid containing cells 10, 11 therein can flow through the microfluidic structure 140 in the direction of flow 141. A retention device in an area of the microfluidic structure can retain cells 10, 11 (target cells and non-target cells) and/or target pathogens 10 contained therein, while the remaining liquid can partially flow in/through the retention device. Furthermore, structures of the cell 10 cell can be labeled by way of binding of a labeling ligand 80, such as a fluorescent labeled antibody and/or a fluorescent labeled DNA stain. Labeled cells 10 (target cells) among the retained cells 10, 11 (target cells and non-target cells) can be detected by way of an image processing device.

[0050] FIG. 3 shows the schematic representation of a recorded image and processing after permeabilization, labeling and/or enrichment in the case of fluorescent labeling and imaging-based detection. The sample containing the cells 10 therein can be irradiated with excitation light 151, for example light having a wavelength in the range of 300 nm to 950 nm of an excitation illumination device 150, such as an LED and/or a laser and/or an illumination source. This excitation illumination can be tailored so as to stimulate the tag 90 to emit fluorescent light 152, such as light having a wavelength in the range of 300 nm to 1000 nm. Fluorescent light 152 can thereupon be absorbed by a means for image processing 160. A magnifying unit 161 and a camera 162 can be assigned to this means for image processing 160. Image data of the camera can be processed and transmitted, preferably wirelessly, to a mobile processor unit 163, such as a smart phone, by way of data transmission 164. The mobile processor unit 163 can evaluate this image data and, furthermore, provide user guidance and control the means for image processing. As a result, present cells and/or pathogens can be localized and displayed in the images. A diagnosis can be established by medical treatment specialists, for example, by transmitting the pathogen images to a processor unit 164, such as another mobile smart phone of such specialists.

[0051] FIG. 4 shows a schematic representation of a microfluidic structure 200, which contains a capillary and/or a microchannel and/or another microfluidic structure, for example. This may contain a sample receptacle 210, in which a volume of a liquid sample is placed. This may be a capillary having a defined volume, for example. The sample receptacle 210 can contain lyophilized substances, such as an anticoagulant. The sample can then through the microfluidic structure 200, for example based on capillary forces. The sample can be divided therein, for example by way of branching of a microchannel, so that a first portion of the sample can flow through a reservoir for a charged, solid substance 220 and can dissolve lyophilized ligands, for example. In a mixing/incubation chamber, the first sample portion can be mixed with this substance or these substances and incubated. The first sample portion can be combined with a second sample portion and be mixed and incubated in a mixing/incubation chamber 240. Capture, enrichment and/or immobilization can take place in the detection area. The means for image processing can detect the target cells/target pathogens in the detection area. The flow process can preferably be maintained by a suction device 250, such as an absorbent non-woven and/or a micropump, but can also be carried out by an external device, such as a pump. The solution can then be collected in a waste reservoir 260. A microfluidic structure, serving as a test strip, can furthermore include a code, such as a bar code, and/or an RFID component for identifying the sample.

Example of the Detection of Malaria Pathogens

[0052] Malaria pathogens undergo several stages during their life cycle. The capillary blood from the finger pad predominantly contains ring-stage plasmodia in infected red blood cells (iRBC). In contrast to the non-infected white blood cells (RBC), the plasmodia contain DNA. A variety of parasitic antigens, such as the PfEMP-1 protein, can be found on the surface of iRBC. For undergoing the immune response, however only one from a pool of many variable, genetically encoded subforms can be found here. So far, no antibodies are known for the pathogen Plasmodium falciparum which could universally detect all variants to as great an extent as possible, and thus be used as a universal antibody for the diagnostic identification of all iRBC infected with Plasmodium falciparum. However, there are several parasitic antigens inside the iRBC containing ring-stage pathogens which do not variably occur in all Plas. falc. Both polyclonal and monoclonal antibodies against plasmodial proteins are commercially available, such as monoclonal mouse IgM antibodies against Plasmodium falciparum of all stages and monoclonal anti-MSP-1 and/or MSP-10 antibodies. Another example of a parasitic antigen is the ring-infected erythrocyte surface antigen (RESA), which, in contrast to its name, cannot be found on the surface of the iRBC, but on the inside of the membrane of infected erythrocytes on components of the iRBC cytoskeleton. Additional antigens, which can be detected by way of specific antibodies, can also be found on the parasitophorous vacuole and, after the Plasmodium membrane has been permeabilized, also in the cytoplasm of the plasmodia. In one embodiment, a defined volume of preferably 1 to 10 m whole blood from the finger pad is applied to the test strip. The erythrocytes are permeabilized and/or lysed by a means for permeabilization, preferably saponin and/or ammonium chloride, so that a labeling ligand, preferably a fluorescent labeled antibody, such as the monoclonal mouse IgM antibody against RBC infected with Plas. falc., Clone III66, labeled with DyLight 488 and/or a fluorescent labeled anti-RESA antibody, can diffuse into the iRBC. There, this binds to the infected RBC and creates an immunofluorescent tag. Alternatively, the parasitic DNA can be labeled using a fluorescent DNA stain, such as Hoechst 33342. A filter now retains all the cells and cell components. The fluorescent tag can be rendered visible by way of the magnifying unit comprising the image processing unit, which serves as a fluorescence microscope. In another embodiment, a magnetic nanoparticle is used as the means for binding, such as a Turbobead PEG streptavidin, to which a biotinylated antibody, such as the monoclonal mouse IgM antibodies against RBC infected with Plas. falc., Clone III66, biotinylated, is bound. Following the permeabilization of the RBC and iRBC, the MNP diffuses into the erythrocytes, where it binds specifically to structures of the infected RBC. A fluorescent DNA stain and/or a fluorescent labeled antibody is added as a second means for labeling, which creates immunofluorescence of the iRBC and/or plasmodia. The iRBC are immobilized and enriched in the detection area by way of a magnet and can be detected by way of fluorescence microscopy using imaging. In a further embodiment, nanoparticles, such as the NanoScreenMag particles from Chemicell GmbH and/or the MyQuVigen nanoparticles from Nvigen, Inc., are added as means for binding and labeling to the permeabilized iRBC. These can be immobilized in the detection area by way of a magnet and detected by the optical magnifying unit by way of fluorescence microscopy using imaging. The image data of the fluorescent labeled iRBC can be subjected to a first processing step by the image processing device, such as a background correction and/or compression. In the next step, a search for objects whose intensity can exceed a critical value and/or which can have a certain size and/or pixel count, and are thus defined as pathogens, can be carried out in the images using an object detection app. This evaluation can be carried out in the image processing device and/or in the mobile processor unit. The object detection can be further validated by appropriate controls, such as cell identification under white light illumination and/or fluorescence identification after membrane staining. A diagnosis of the patient can be inferred from the detection of pathogens.

[0053] Another possible application example for the detection of malaria pathogens is the addition of saponin as a means for lysis and permeabilization to erythrocytes infected with plasmodia in a whole blood sample. Under the action of saponin, the outer erythrocyte membrane is lysed, and the parasite can be released therefrom, wherein the parasitophorous vacuole is preserved. At least one structure, such as parasitophorous antigens, for example the proteins RAP1 or EXP2, can be found on this parasitophorous vacuole. These are stored in rhoptries, micronemes or dense granules, for example, and are thus already present even in early-stage trophozoites. At least one capture ligand 60 can bind to these, and in particular at least one antibody against plasmodial proteins such as RAP1 or EXP2, for example the monoclonal antibody anti-RAP-1, Clone 2.29, or the monoclonal antibody anti-EXP-2, Clone 2.2. This antibody is preferably bound to at least one magnetic microparticle or nanoparticle 70, and in particular to a Dynabead or Dynabead Myone, and/or at least one or more labeling ligands 80, preferably at least one or more labeled antibodies, such as the monoclonal antibody anti-RAP-1, Clone 2.29, or the monoclonal antibody anti-EXP-2, Clone 2.2, against plasmodial proteins such as RAP1 or EXP2. These antibodies can preferably be labeled with a spectrally distinguishable fluorescent dye, such as DY-396XL from Dyomics GmbH, or quantum dots, such as Qdots from Thermo Fisher Scientific or Candots from Candots GmbH. Moreover, at least one (preferably fluorescent) DNA stain can be used against plasmodial DNA, so as to detect, in particular, at least one structure 40, 50, 20 of plasmodia 20 and/or of erythrocytes 10 infected with at least one or more plasmodia 20, and/or to capture at least one infected erythrocyte 10 and/or at least one structure 40, 50, 20 of plasmodia by way of retention and/or magnetic separation in a means for localization, immobilization and/or enrichment 120, such as a microtiter plate having at least 96, and in particular 1536 wells, a microcuvette, a filter structure without or coated with capture ligands or a fluidic structure.

Example of the Detection of Sepsis Pathogens

[0054] Sepsis strikes approximately 18 million patients each year and is fatal in approximately 50% of cases. More than 25 different pathogens can cause sepsis, such as bacteria (meningcocci, streptococci and the like) and/or fungal infections. Commercial rapid tests already exist for streptococci, for example. However, these are usually composed of multiple steps, since first the antigen has to be isolated from the cell wall using multiple reagents and then be detected in an indicator reaction. The high specificity of 95 to 100% is well-documented. However, the sensitivity of the tests is only 70 to 90%, which means that the result is a false negative in 10 to 30%. Infectious bacteria are generally accompanied by a regional and temporal change of the surface antigens. A rapid test according to the invention can be carried out by binding and labeling other antigens, to which ligands would not have access without permeabilization of the cells. Comparable to the detection based on the example of a malaria pathogen, different embodiments exist, such as A) retaining all cells and immunofluorescence detection by way of a fluorescent labeled antibody against an internal, more preserved antigen; B) magnetic capturing and simultaneous fluorescence labeling by way of a fluorescent MNP to which a specific antibody is coupled; and/or C) magnetic capturing by way of an MNP, preferably a Turbobead, using a specific antibody and fluorescent labeling by way of a second fluorescent labeled antibody and/or a DNA stain.

[0055] Example of an application of the invention for the analysis of food and/or drinking water: detection of legionella in drinking water

[0056] Legionella are movable rod-shaped bacteria having an average length of 2 to 5 m and a diameter of 0.5 to 0.8 m. They are common in surface water and in drinking water lines in a number of species and serogroups. An infection with Legionella pneumophila occurs through atomized water or mist and can cause what is known as Legionnaires' disease, a severe case of pneumonia that is fatal in 10 to 15% of cases. Commercial rapid tests usually include a sampling device, which is mailed to a laboratory, where it is analyzed by way of a bacteria culture in agar plates within approximately 10 days. One embodiment of the invention can include a test strip, for example, which is designed to filter a volume of drinking water in the range of milliliters to several liters and/or cubic meters, so that all cells, bacteria and pathogens contained therein are retained and situated in the detection area. This can be done, for example, using a microporous filtration membrane having a pore size of 0.45 m. Afterwards, a means for permeabilizing the pathogens is introduced, for example from a blister contained in the test strip, so that the filtration membrane is incubated therewith. Moreover, for example, a fluorescent labeled antibody and/or fluorescent nanoparticle including an antibody are added, which binds specifically to antigens and/or cellular structures of legionella, to which no access would exist without means for permeabilization and which have a locally and temporally substantially constant structure. The pathogens can be detected by way of immunofluorescence by the magnifying unit comprising the image processing device using imaging. In another embodiment, first a volume of drinking water in the range of milliliters to several liters and/or cubic meters is mixed in a mixing chamber with a means for permeabilization and magnetic microparticles, such as Dynabeads, or nanoparticles, such as Turbobeads, to which a specific antibody against antigens of legionella is coupled. After incubation, the mixture is conducted in a test strip past a magnet in a detection area. The MNPs are captured from the solution, whereby bound legionella and/or the components thereof are immobilized. The fluorescent staining can be carried out using a labeling ligand, such as a fluorescent DNA stain and/or a fluorescent labeled antibody and/or a fluorescent nanoparticle including a bound antibody. The detection can again take place by way of imaging immunofluorescence.

[0057] As an extension of this example of an application, a sample, in particular of a body fluid such as urine and/or stool, and/or a food sample and/or a drinking water sample can be used, which can be mixed with a buffer to dissolve the same. Solid components can subsequently be separated, for example by way of filtration, sedimentation and/or centrifugation. Pathogens such as legionella and/or eggs of the pathogens, such as of Ascaris, Trichiuris, Amylostoma, Taenia worms, can be lysed and/or permeabilized using a means for lysis and/or permeabilization 110. The pathogens, eggs of the pathogens and/or the components thereof can subsequently be labeled using a labeling ligand 80 and/or be enriched by way of a means for localization, immobilization and/or enrichment 120, such as a filter structure and/or magnetic particles, and/or be detected by way of imaging microscopy.

[0058] The above description of the embodiments describes the present invention exclusively based on examples. Individual features of the embodiments, provided they are technically expedient, can, of course, be freely combined with each other without departing from the scope of the present invention.

LIST OF REFERENCE NUMERALS

[0059] 10 target cell or target pathogen [0060] 11 cell [0061] 20 structure of a cell and/or of a pathogen [0062] 30 structure for compartmentalization [0063] 31 opening generated in 30 and/or receiving mechanism through 30 [0064] 40 target structure [0065] 50 target structure [0066] 60 capture ligand [0067] 70 magnetic nanoparticle [0068] 80 labeling ligand [0069] 90 tag [0070] 110 means for permeabilization [0071] 120 means for localization, immobilization and/or enrichment [0072] 121 reaction vessel [0073] 122 retention device [0074] 130 magnet [0075] 131 magnetic attractive force [0076] 140 microfluidic structure [0077] 141 direction of flow [0078] 150 illumination unit [0079] 151 excitation light [0080] 152 fluorescent light [0081] 160 means for image processing [0082] 161 magnifying unit [0083] 162 camera [0084] 163 mobile processor unit [0085] 164 data transmission [0086] 165 mobile processor unit [0087] 200 microfluidic structure [0088] 210 sample receptacle [0089] 220 reservoir [0090] 230 blister reservoir [0091] 240 mixer structure, incubation structure [0092] 250 suction device [0093] 260 waste reservoir [0094] 270 code [0095] 280 detection area