EP3479122A1 - Test rapide pour le dépistage d'agents pathogènes et de cellules et procédé correspondant - Google Patents

Test rapide pour le dépistage d'agents pathogènes et de cellules et procédé correspondant

Info

Publication number
EP3479122A1
EP3479122A1 EP17737240.6A EP17737240A EP3479122A1 EP 3479122 A1 EP3479122 A1 EP 3479122A1 EP 17737240 A EP17737240 A EP 17737240A EP 3479122 A1 EP3479122 A1 EP 3479122A1
Authority
EP
European Patent Office
Prior art keywords
pathogens
immobilization
enrichment
localization
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17737240.6A
Other languages
German (de)
English (en)
Inventor
Guido BÖSE
Markus Ganter
Taleieh Rajabi
Andreas Guber
Ralf Ahrens
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zendia GmbH
Original Assignee
Zendia GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zendia GmbH filed Critical Zendia GmbH
Publication of EP3479122A1 publication Critical patent/EP3479122A1/fr
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
    • G01N2333/4353—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from nematodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/445—Plasmodium

Definitions

  • the present invention relates to a test system for the diagnosis of diseases according to the independent claim 1 and a test method for the diagnosis of diseases according to the independent claim 8 and a use of a test system and / or test method for the diagnosis of diseases according to the independent claim 15.
  • Test systems for the diagnosis of Diseases are known in the art. For example, a variety of analytical methods and methods are used to elicit antibody reactions, so-called immune reactions, which are used for the determination of (bio) markers and many other substances / analytes. Furthermore, microscopy-based methods in the field of diagnostics are described.
  • PoC testing procedures are diagnostic examinations that can not be performed directly and individually on a patient / subject within a short time on-site.
  • PoC test systems such as Immunochromatographic test strips which detect a soluble biomolecule, eg a hormone or a protein in blood, urine or saliva, by means of a color reaction via antibody binding
  • Known PoC test strips are for example pregnancy tests, blood coagulation tests, offered with or without a measuring device Rapid test methods such as the lateral flow test (LFT), flow-through test (FTT), agglutination test (AT) or solid-phase test (SPT) are all known for rapid detection of analytes suitable for visual evaluation in the PoC range
  • LFT lateral flow test
  • FTT flow-through test
  • AT agglutination test
  • SPT solid-phase test
  • a small amount of blood is taken, of which a smear or preparation is prepared as a so-called thick drop and stained with Giemsa dye.
  • Giemsa dye After fixation and drying of the preparation of the parasite can be detected under the microscope in the red blood cells (erythrocytes) and differentiated by the expert.
  • This method requires not only a trained practitioner, but also a laboratory environment and an expensive microscope. In most malaria areas, such a laboratory is not available anyway. Existing molecular biology tests are also expensive and also require deeper user knowledge. Also known are lateral flow rapid tests (supra), which can detect parasite-specific antigens, but not the parasites as such or affected cells and therefore do not allow pathogen or cell diagnostics.
  • a robust and rapid diagnosis for the rapid detection of pathogens at the site of the sample name is needed to determine the source of infection, to initiate appropriate therapy of sick patients and to prevent further infection of humans.
  • Immune detection of the pathogens in rapid tests such as the lateral flow test (LFT), flow-through test (FTT), agglutination test (AT) or solid-phase test (SPT). Therefore, those systems of particular interest are those which have surface antigen-independent analysis of the agents and cells and can be used as POC and / or rapid tests. Detection method via DNA or RNA analysis e.g. By PCR or cultivation of the pathogens are complex in terms of apparatus and time and costly. Thus, there is a great need to develop a test system with a surface antigen-independent analysis as POC and / or rapid tests, which allows complex diagnostics. The complex diagnostics should in particular enable a pathogen and cell diagnostics from body fluids or food and / or drinking water.
  • LFT lateral flow test
  • FTT flow-through test
  • AT agglutination test
  • SPT solid-phase test
  • test system and a test method and a use of the test system and / or test method which at least partially remedy the aforementioned disadvantages.
  • object of the present invention to detect inexpensive and in a very short time a variety of pathogens and / or cells, in particular independent of a laboratory equipment.
  • a test system for the diagnosis of diseases with the technical features of claim 1 and a test method for the diagnosis of diseases with the features of claim 8 and a use of the test system and / or test method with the features of claim 15 is proposed, the following particular importance.
  • the test system comprises at least one means for permeabilization and / or lysis of at least one pathogen and / or at least one cell and / or a means for binding and / or marking of parts of the pathogens and / or cells and / or a means for localization, Immobilization and / or enrichment of at least one component of a pathogen and / or a cell and / or a means for image processing, whereby an optical reading of at least one means for localization, immobilization and / or enrichment is feasible.
  • the readout can take place via an arranged optical magnification unit.
  • the reading can take place via a mobile computer unit.
  • structures for means for binding and / or labeling can preferably be made available to a fluorescent label, which without these means would not be accessible by compartmentalizing structures such as a lipid membrane or cell wall.
  • immobilization and / or enrichment parts of the pathogens and / or cells can be arranged from a liquid sample.
  • the marking can be optically read with the aid of a means for image processing, and a sufficient intensity and contrast effect can be obtained for one or more images and / or images which can be read out via the optical magnification unit and image processing device in the mobile computer unit, so that effective image processing can be done and thus the desired pathogens and Cells can be detected by imaging.
  • a specific immunostaining and / or specific DNA staining infected cells appear in contrast as colored and / or fluorescent dots and / or areas in front of a less colored and / or weaker fluorescent background, while uninfected cells may be invisible and / or only weak can remain visible.
  • an optical magnification unit may constitute a unit which may include at least one objective and / or may include an array of one or more objectives and / or lenses, similar to a microscope, which may have sufficient magnification. Such an enlargement can be achieved, for example, via a connection with a microscope. If such a microscope is used, image data can alternatively be transmitted directly to the computer unit.
  • powers of the optical magnification unit in the presence of the image processing device are preferably conceivable with magnifications of approximately 2.5 times to approximately 5 times, preferably approximately 10 times to approximately 1000 times.
  • the resolution can be about 5 ⁇ , preferably about 0.1 ⁇ to about 2 ⁇ , in particular of about less than 0.5 ⁇ .
  • the magnification factor can be fixed or variable.
  • a variable lens such as a liquid lens, for example, the company Optotune Switzerland AG or Vario Optics AG can be used.
  • the optical magnification unit or image processing device may preferably have one or more arbitrary light sources, such as a white light and / or fluorescence illumination.
  • the fluorescence illumination can be used as excitation light for fluorescent dyes.
  • an image processing device according to the invention can be arranged to the mobile computer unit, in particular in the form of a camera.
  • the image processing device may be used to acquire image data from an optical magnification unit.
  • a mobile computer unit can represent a unit that is made available, for example, by a mobile telephone with computer function (smartphone) and / or by a laptop and / or tablet computer (tablet, tablet computer).
  • the mobile computer unit can essentially comprise a central unit and / or a processor which can execute the arithmetic and process steps required for image processing.
  • the image processing may further comprise an analysis mode, wherein summary gray levels, color values and / or color contrasts, preferably fluorescence signals, can be detected in an image detail.
  • the detection can be qualitative and / or quantitative.
  • This critical value is determined empirically for the test system and serves, for example, to distinguish it from the background signal of the marker ligand of specifically bound labeling ligand still present in the solution in an immunofluorescence.
  • the quality of the image processing can be increased by successively examining individual image sections of a larger field. This can be done by a mere selection in image processing. Alternatively or additionally, the quality of the image processing can also be done by an optical magnification of each image section, for example, a delineated object with an intensity above a critical value can be defined as a pathogen.
  • a quality analysis in the context of the image analysis.
  • the standard deviation of the results can be calculated in several sections. Alternatively or additionally, it can also be determined how close a calculated value is to a critical value. A close proximity of the calculated value to the critical value or to a large standard deviation can be concluded from the poor quality of the measurement.
  • a program can thus likewise be carried out on a computer unit, for example as an app on a smartphone, which has at least one of the following basic functions: reading of image data, image processing, quantitative and / or qualitative output of an image processing and / or analysis result.
  • the program may include a quality analysis related to the quality of the analysis of a sample.
  • the link with further information comes into consideration, in particular internal or external databases on clinical pictures, addresses of medical services, transmission of the pathogen images to healthcare professionals and the like.
  • the test system comprises means for localization, immobilization and / or enrichment elements for receiving a sample fluid.
  • the sample fluid originates from a body fluid and / or a food sample and / or drinking water.
  • a body fluid may preferably be blood, whole blood, urine, saliva, synovial fluid, cerebrospinal fluid, plasma and / or serum and / or tear fluid, sweat, lymph fluid and / or intercellular fluid.
  • Including conventional adjuvants and additives such as anticoagulants, protease inhibitors, stabilizers and / or enzyme inhibitors are treated.
  • Patients are each subject, regardless of a Symptomatikund / or disease and that human and animal, especially mammal.
  • Such body fluids contain analytes, particularly cells that may contain infected cells and / or pathogens as such.
  • pathogens are bacteria, fungi, viruses and / or parasites.
  • a sample fluid of a food sample may comprise any dilution, solution, extract and / or smear of a foodstuff.
  • a sample fluid of drinking water may further comprise an unchanged and / or concentrated and / or diluted sample and / or a liquid uptake of a filtration residue.
  • test system has means for permeabilization, wherein advantageously access for ligands to at least one biomolecule and / or to at least one structure of a pathogen and / or to at least one cell can be achieved.
  • structure and / or the cell it is conceivable for the structure and / or the cell to have at least one opening with a size between approximately 1 nm and approximately 12 ⁇ m can have. Complete lysis of the cell can also be produced.
  • a permeabilization and / or lysis of pathogens and / or cells may be a process for generating a temporary and / or permanent permeability of outer and / or inner cell membranes, cell walls, murein envelopes, virus envelope and / or lipid membranes and / or other compartmentalizing structures, to allow accessibility of antibodies, labeled antibodies, ligands and / or ligand-loaded magnetic particles to internal structures, antigens and / or biomolecules of the pathogens and / or cells.
  • a means for permeabilizing and / or lysing pathogens and / or cells may comprise a moiety and / or substance that provides accessibility to biomolecules and / or structures of the pathogens and / or cells that are typically present, for example, in living pathogens and / or or cells are separated by a lipid membrane, cell wall, virus envelope and / or other Zellumhüllenden layer and are not accessible for example for immunoanalysis.
  • Such agent may provide pores in a membrane by pore proteins and / or destabilize the membrane by cholesterol withdrawal and create openings in the membrane and / or by osmotic effects such as swelling and bursting of the cells lead to openings in the cell membrane and / or by electrical and / or generate mechanical pulses openings.
  • An agent may further comprise any other agents which provide one or more openings in the cell membrane, cell wall, viral envelope, lipid membranes and / or penetration of these, for example by similar means of transfection applied agents such as fused and / or imported vesicles, magnetic particles and / or allow penetrating nanoparticles.
  • These agents can be substances for permeabilization and / or lysis, such as saponins, for example for the lysis of erythrocytes, bacteria, fungal cells and / or other eukaryotic cells, digitonin for the temporary permeabilization of, for example, eukaryotic cells, porins, for example for the permeabilization of lipid membranes, streptolysin O.
  • ammonium chloride eg for the lysis of erythrocytes
  • detergents such as deoxycholates, Triton X100, NP-40 and / or other substances such as acetone and / or ethanol for destabilization of the cell membrane, ethylenediaminetetraacetic acid for permeabilization of the outer membrane of gram-negative bacteria, lysozyme and / or Autolysins eg for lysis of the peptidoglycanic wall of gram-positive bacteria, lactoferrin, defensins and cathelicidins of gram-negative bacteria and other substances, but also an electric field which, like an electroporation, can make the cell membrane temporarily and / or permanently permeable and / or a mechanical and / or other action which leads to permeabilization and / or lysis and provides extended accessibility of biomolecules of the pathogens and / or cells for ligands.
  • These agents can be combined with fixing substances such as paraformaldehyde, glutaraldehyde, acetone, methanol, ethanol.
  • the sizes of the apertures produced can range from about 1 nm to about 12 ⁇ , and can result in temporary permeabilization for a few milliseconds until permanent lysis of the cells.
  • means may be conceivable which can serve to accommodate ligands and / or magnetic particles without the formation of openings, such as means which can also be used in a transfection, such as vesicles, magnetic particles, penetrating nanoparticles, import-triggering substances.
  • they may be agents which lead to a release of biological structures and / or molecules and / or other parts of the pathogens and / or cells, which would remain without these agents inside the cells and / or pathogens and thus before a Detection by the immune system would be shielded.
  • This may include lysis and / or other destruction of the pathogens and / or cells and / or the induced release and / or release of biological structures and / or molecules by export and / or through pores and / or openings.
  • means for the localization, immobilization and / or enrichment of at least one component of pathogens and / or cells can be used for the test system.
  • at least one component of pathogens and / or cells can be enriched.
  • the means for localization, immobilization and / or enrichment may be a reaction vessel such as an Eppendorf reaction vessel and / or a filtration column.
  • a sample and / or means for permeabilization and / or means for binding and / or means for labeling may be miscible with each other and may for example be immobilized via a magnet and detected via a means for image processing such as a fluorescence spectrometer and / or a fluorescence imaging microscope become.
  • the means for localization, immobilization and / or enrichment may have a microfluidic structure for dissolving and / or mixing substances.
  • Such means for localization, immobilization and / or enrichment may preferably be a test strip having a microfluidic structure.
  • Such a test strip is preferably made of one or more transparent material and / or materials, such as a planar plastic and / or glass, that may be capable of receiving a body fluid and / or other fluid sample.
  • a sample can pass through the test strip, in particular by means of at least one incorporated and / or applied channel and / or microchannel, in particular with a rectangular, trapezoidal and / or arcuate cross section.
  • the channel may be a capillary and / or permit laminar and / or non-laminar flow by gravity and / or capillary force and / or pumping forces and / or other forces.
  • the test strip may preferably be used to carry out the detection method of pathogens and / or cells consisting of a sample uptake, addition and mixing with one or more permeabilization agents, addition and mixing with one or more binding and / or marking structures of the Pathogens, cells and / or their components, an incubation of the sample, a subsequent localization, immobilization and / or enrichment of the pathogens and / or cells and / or their components in a detection range include.
  • the test strip may also preferably provide a flow maintenance device, for larger volumes up to several liters, to external pumping systems.
  • the sample receptacle can have a reservoir into which a defined volume, in particular of a few microliters of a sample, for example with a micropipette, can be added. For volumes of several liters, these can be given up via a connection from an external system.
  • the sample can be provided with (dried) chemicals such as EDTA, heparin for anticoagulation and / or dyes for histological cell staining, which can be solved by the discontinued sample.
  • the uptake can preferably take place by means of a capillary with a defined volume and a filling indication.
  • the whole blood can preferably be sucked in until full filling and thus provide a defined volume.
  • the capillary is located, for example, in a lid that fits the test strip, then the defined blood volume can then be applied to the test strip by attaching the lid to the test strip.
  • the lid may preferably include structures that irreversibly engage when attached to the test strip.
  • Another embodiment may have a microfluidic structure with defined Volume belong to the test strip include, in which the whole blood is sucked in and can get in contact with the other microfluidic system, for example, by moving the capillary and / or microvalves and / or other devices after the volume definition, so that the whole blood are applied in this can.
  • the application of the agent for permeabilization and / or lysis and the means for binding and / or for marking specific structures can be carried out via a solution of lyophilized substances by the passing sample.
  • the anterior volume in the test strip may comprise more solute than the posterior volume.
  • the test strip may preferably have at least one mixer structure which can mix the dissolved substances with the sample. In the case of a microfluidic structure, this may be a mixing chamber with and / or without external mechanical action, a zigzag structure and / or another device for mixing.
  • a further embodiment may comprise a splitting of the microfluidic sample flow into at least two channels, it being possible for the lyophilized substances to be added to a sample part in a first channel and for a sample part to be conducted past the latter in the second channel. Subsequently, the channels can be at least partially brought together again and mixed in a mixer structure, the two sample parts together.
  • the flow rates may preferably be determined by the channel dimensions. With such an embodiment it can be achieved that the rear sample volume from the second channel can also be mixed with sample volume containing substances from the first channel. In this case, an immunofluorescence staining with a labeling ligand can take place simultaneously.
  • test strip may preferably provide a flow maintenance device for small volumes of a few microliters, for larger volumes up to several liters connections to external pumping systems.
  • the device for maintaining the microfluidic flow can preferably comprise a capillary pump following the detection region, which can be activated by capillary forces Draws liquid into a waste compartment subdivided with microstructures.
  • a material which exerts a suction force such as a filter fleece and / or absorbent fleece, may be integrated into the test strip such that it can at least temporarily make contact with the microfluidic channel and preferentially draw liquid through the test strip.
  • a micropump can be integrated into the test strip, which can control the flow. This can be a stamp which presses liquid from a blister into the microfluidic channel, a suction device which sucks liquid from the channel via negative pressure and / or another device such as, for example, the micropump mp6 from Bartels Mikrotechnik GmbH.
  • a barcode can be printed on a test strip and / or an RFID element can be incorporated, which may contain usual information about the test strip, eg the type of test (eg diagnostics for malarial pathogens, Legionella and / or sepsis) and / or Expiration date of the test strip, among others
  • the test system may comprise means for localization, immobilization and / or enrichment, which on the one hand can have a microfluidic structure for incubating a sample fluid with capture and / or label ligand and at least one detection region.
  • at least one constituent of at least one labeled pathogen and / or at least one labeled cell can be accommodated on the means for localization, immobilization and / or enrichment.
  • the staining may preferably be a simple histological staining of the cell plasma, a lipid membrane, cell wall and / or the DNA and / or an immunochemical stain such as a fluorescent stain.
  • the background signal of the specific marking by the dyeing solution remaining in the channel may not be relevant in the measurement since the dyeing solution may preferably be low concentrated.
  • the dyeing liquid spreads in the channel, and / or has flowed through it, one or more unbound label ligands may be distributed in the channel which may bind to permeabilizable markers of the pathogens and / or cells.
  • the concentration of labeling ligands in the dyeing liquid is preferably selected so that label ligands can be enriched in the infected cells, pathogens and / or their components against the free label ligands present in the liquid.
  • pathogens, cells, infected cells and / or components thereof may preferably appear in contrast as lighter colored / fluorescent dots and / or areas in front of a colored / fluorescent background, while other liquid components such as uninfected cells may remain invisible and / or only weakly visible .
  • Incubation of the sample can be achieved, for example, by an embodiment of the test strip which may contain an incubation structure. This can be, for example, a zigzag structure, which can be flowed through in periods of a few seconds to several minutes and can both mix the sample and allow incubation.
  • substances for permeabilization and / or lysis of the pathogens and / or cells can be presented in a lyophilized form and, as described, dissolved through part of the sample volume, mixed into the sample and incubated. If the pathogens and / or cells are subsequently nonspecifically or specifically retained, immobilized and / or trapped in the microfluidic structure, sequential addition of the labeling ligand to the marking of the structures sought can also take place. In one embodiment, a wash and / or a scavenger ligand and / or a label ligand can be added externally.
  • the test strip may comprise a fluid-filled blister which may be opened by the user and / or a device such as a mechanical punch in the means for receiving the test tire such as an optical reader.
  • the liquid can enter the microfluidic channel and can either already contain the ligands and / or be dissolved in the channel from an initially presented, lyophilized form and subsequently surround the pathogens and cells.
  • Such a test strip may preferably have a detection region of the microfluidic channel system in which preferably fluorescently labeled pathogens, cells and / or their components are immobilized and preferably can be enriched by holding them while the remaining liquid can continue to flow to a collection reservoir ,
  • the detection area can be arbitrarily large and arbitrarily designed configured.
  • test strip may contain a means for localization, immobilization and / or enrichment of at least one microfluidic structure made of PET, in which an aqueous liquid preferably passively flows through the material properties of the PET without further coating.
  • This microfluidic structure can be completely and / or partially closed with a film instead of a rigid lid.
  • the structure can be subdivided into one or more areas, which are first closed, thus forming a dead end, as a result of which the liquid does not flow further through it and which liquid can flow through the liquid after opening, such as, for example, piercing a film over the ventilation opening located behind it.
  • This can in particular form a subdivision into a sample receiving area, a mixing area and a detection area. In the sample receiving area, a defined sample volume of a liquid, such as whole blood from the fingertip, may be picked up due to capillary forces.
  • a mixing area can be arranged, behind which a second ventilation hole can be arranged, which is initially closed. If the film is opened over this and pierced, for example, the sample flows into the mixing area in which the sample is preferably with lyophilized substances such as an anticoagulant and / or a capture ligand with or without Magnetbeads and / or one or more labeling ligands and / or Stabilizers and / or a means for lysis and / or permeabilization can be mixed.
  • lyophilized substances such as an anticoagulant and / or a capture ligand with or without Magnetbeads and / or one or more labeling ligands and / or Stabilizers and / or a means for lysis and / or permeabilization can be mixed.
  • the sample receiving area and the mixing area can also be overlapping or be identical.
  • a detection area can be arranged, which provides a viewing window for an imaging microscopic measurement. If another ventilation hole located behind the detection area, for example, closed with foil, is opened, the sample flows into the detection area.
  • the pathogens can be localized and / or enriched and / or, preferably by an external magnetic field, immobilized and imaged.
  • the test system may also comprise means for binding at least one capture ligand and / or at least one labeling ligand for labeling, in particular staining.
  • an aptamer and / or a ligand can be included and / or a micro-particle, nanoparticles, magnetic nanoparticles can be included, which can be coupled with an antibody, aptamer, ligands.
  • an at least magnetic nanoparticle preferably a turbobead and / or a metal core, may be bindable to at least one constituent of pathogens and / or cells via a coupled ligand.
  • Binders and / or markers may comprise one or more capture ligands and / or label ligands which may preferentially bind to antigens and / or biomolecules of whole or part of cells and / or agents.
  • Ligands may preferably be antibodies, aptamers, peptides, protein ligands, micro-particles, nano-particles, magnetic beads, reactive substances, but also other ligands and / or a combination of the abovementioned, which bind to so-called markers such as antigens, biomolecules and / or other structures of the pathogens and / or cells and / or their constituents can specifically bind.
  • ligands can first be prepared by a means for permeabilization with markers such as biomolecules, structures and / or antigens of the pathogens and / or cells of which they are without the means for permeabilization by a cell envelope, cell membrane, cell wall, virus envelope, lipid membrane and / or other compartmentalized structure would come into contact.
  • Capture ligands may serve to immobilize the pathogens, cells and / or their constituents in order to preferably achieve an enrichment over a detection range.
  • magnetic particles such as preferably magnetic microparticles, in particular Dynabeads or magnetic nanoparticles (MNP), in particular turbobeads, but also other non-fluorescent and / or fluorescent MNP, with or without tethered Antibodies, aptamers and / or ligands
  • MNP magnetic nanoparticles
  • this may mean the immobilization of bound pathogens, cells and / or their constituents, preferably on the bottom and / or the ceiling of a microchannel, over a detection area by means of a magnet.
  • label ligands can be used which can preferentially bind to a second, independent binding site of pathogens, cells and / or their constituents.
  • ligands may preferably be coupled with a fluorescent dye, fluorescent nanoparticles such as quantum dots and / or dye, magnetic particles and / or gold particles, so that their binding to the cell surface produces a sufficient signal, preferably a fluorescence signal, and preferably by means of an optical signal Magnification can be made visible against the background, so that individual visual color values, grayscale and / or contrasts can arise. Therefore, the invention may also relate to a test system, wherein the analyte bound to the test strip, cells, pathogens are labeled by means of a labeling ligand, which is coupled to a dye preferably a fluorescent dye, dye, nanoparticles, magnetic particles and / or gold particles, and preferably an image analysis can be made accessible.
  • a labeling ligand which is coupled to a dye preferably a fluorescent dye, dye, nanoparticles, magnetic particles and / or gold particles, and preferably an image analysis can be made accessible.
  • a double detection in the sandwich system on the one hand for example by a capture ligand of specific antibody to magnetic particles and on the other hand by a labeling ligand such as a fluorescently labeled antibody take place.
  • a capture ligand of specific antibody to magnetic particles and on the other hand by a labeling ligand such as a fluorescently labeled antibody take place.
  • a labeling ligand such as a fluorescently labeled antibody
  • the method can be the enrichment and clear visual recognition of cell types such as malaria-infected erythrocytes, CD4 + cells and / or also infected cells in HIV diagnostics, also the detection and typing of bacteria in the diagnosis of eg sepsis, Legionella, cholera, Allow tuberculosis etc.
  • a labeling ligand also coupled enzymes can be used for an enzymatic detection reaction, so that a color reaction and / or electrochemical reaction can be detected, which is detectable.
  • a filtration device for example consisting of a filter material such as For example, a "Vivid" plasma separation membrane from Pall Corp., a microporous and / or nanoporous membrane with subcellular pore diameters, and / or other structures having a sifting function may permit passage of the fluid, but nonspecific pathogens and / or cells This may be integrated at one end of the channel and / or laterally in a channel wall.
  • the accumulated pathogens, cells, infected cells and / or their constituents may be specifically marked by one or more labeling ligands
  • labeling ligands such as fluorescently labeled antibodies, aptamers and / or other ligands may be a specific coloration of the sought structures of the pathogens, cells and / or their constituents.
  • the test strip may contain a filter for nonspecific damming all Cells and / or pathogens while the labeling of the desired pathogens can preferably
  • fluorescence staining of the DNA can also take place.
  • parallel membrane staining can be carried out to detect all cell outlines by means of white light and / or via spectrally separated fluorescence detection.
  • magnetic particles can preferably be coupled to the ligands which can specifically bind to the cellular structures, thus enabling their magnetic capture and accumulation by application of an external magnetic field.
  • the magnetic force may preferably lead to immobilization of the magnetic particles in the test strip, such as on the floor and / or on the ceiling of a microfluidic channel.
  • Commercial magnetic particles which can usually be used for catching cells via surface antigens by means of antibodies, aptamers and / or ligands, may in particular have a diameter of approximately 1 nm to approximately 12 ⁇ m.
  • Thermo Fisher Scientific offers, for example, Dynabeads 1 ⁇ m micron microparticles or 2.8 ⁇ m Dynebeads with various surface groups such as streptavidin, biotin and / or chemical coupling groups such as azide and / or Amino groups for attachment of biomolecules, antibodies, aptamers and / or ligands.
  • microparticles may be too large to be able to penetrate through the openings produced into permeabilized pathogens and / or cells and / or be absorbed by other techniques. This can for example take place only when the pathogens and / or cells are lysed and fragmented, so that structures from the inside can be accessible to the microparticles.
  • the pathogens and / or cells may be permeabilized but maintained in their form by the cytoskeleton and thus remain physiologically recognizable.
  • Magnetic nanoparticles (MNP) may have a diameter of about 5 nm to about 500 nm, and thus penetrate through openings in permeabilized pathogens and / or cells.
  • non-fluorescent MNPs with coupled antibodies, aptamers and / or ligands can be used in order to be able to specifically bind to antigens and / or structures after permeabilization of the desired pathogens and / or cells.
  • the MNPs with the bound pathogens, cells and / or their components can be immobilized in the detection area, for example at the channel ceiling and / or the channel bottom.
  • a labeling ligand a specific, detectable signal can be generated on the desired pathogens, cells and / or their constituents.
  • immunofluorescence can be produced in the sandwich structure.
  • the DNA can be stained by plasmodia using a fluorescent and / or non-fluorescent DNA dye.
  • the Magvigen particles from Nvigen Inc. have diameters of, for example, 200 nm to 500 nm and are available on their surface with coupling groups and / or proteins.
  • a chemical coupling can be possible as well as existing on the MNP proteins such as avidin and / or a second antibody.
  • Turbobeads Llc. also offers magnetic nanoparticles, called turbobeads, with various surface groups such as streptavidin, biotin and / or chemical coupling groups such as azide and / or amino groups for attachment of biomolecules, antibodies, aptamers and / or ligands.
  • Turbobeads can have a diameter of about 30 nm, but preferably about three times stronger magnetic properties by using a metallic core instead of the usual MNP ferrite core. The magnetic separation can thus be significantly faster and more efficient than with conventional MNPs.
  • Magnetic nanoparticles which are fluorescent at the same time, can bind the target structures via coupled antibodies, aptamers and / or ligands and can be used both for magnetic capture and for fluorescence labeling.
  • the magnetic and fluorescent MyQuVigen particles from Nvigen Inc. and also the NanoScreenMag MNP from Chemicell GmbH are also offered with different surface groups, eg for antibody coupling. This combination may have the advantage of a simple method of simultaneously immobilizing the labeled pathogens over a magnet and the fluorescent label.
  • a fluorescence background can arise from single, unbound particles, which must be reliably differentiated from pathogen-bound MNPs.
  • the immobilization of the pathogens, cells and / or their components can be separated from the label, that is, separate capture ligands and labeling ligands can be used.
  • the test method for diagnosing diseases comprises at least one of the following steps: applying a sample fluid, in particular a liquid, to the means for localization, immobilization and / or enrichment.
  • a sample fluid in particular a liquid
  • this may allow application at the point-of-care, for example, simply by sucking a drop of capillary blood from the fingertip by an untrained person and automated processing of the sample in a sealed means for localization, immobilization and / or enrichment without further intervention by the user and / or laboratory equipment, for example, for the precise addition of substances, pathogen culture, centrifugation, etc. is necessary.
  • a means for permeabilization to the means for localization, immobilization and / or enrichment can be carried out, whereby access of ligands to structures of at least one exciter contained in the sample fluid and / or at least one cell is achieved becomes.
  • This can preferably be done by dissolving a substance lyophilised in the means for localization, immobilization and / or enrichment and / or by opening a blister with the substance presented without user intervention.
  • the means for localization, immobilization and / or enrichment may provide the necessary mixing and incubation of the sample with the means for permeabilization. This can lead to permeabilization and / or lysis of compartmentalizing components of the pathogens and / or cells such as a lipid membrane and / or cell wall.
  • a labeling ligand in particular a fluorescent dye to at least one antibody and / or a DNA dye and / or applying and / or releasing a capture ligand to the means for localization, immobilization and / or enrichment be performed.
  • a labeling ligand in particular a fluorescent dye to at least one antibody and / or a DNA dye and / or applying and / or releasing a capture ligand to the means for localization, immobilization and / or enrichment be performed.
  • This may preferably be done without user intervention by dissolving a substance lyophilised in the means for localization, immobilization and / or enrichment and / or by opening a blister with the substance presented.
  • the means for localization, immobilization and / or enrichment can thereby provide the necessary mixing and incubation of the sample with the means for binding and the means for labeling.
  • marking at least one component of at least one pathogen and / or at least one cell is conceivable.
  • a means for binding and / or labeling can gain access to structures of the pathogen and / or cells, for example inside the cell, so that specific binding of, for example, antibodies to antigens can take place.
  • fluorescently labeled antibodies this can produce a specific immunostaining.
  • the enrichment of at least one component of an exciter and / or a cell in particular by means of damming and / or magnetic separation in a means for localization, immobilization and / or enrichment is conceivable.
  • magnetic nanoparticles When using, for example, magnetic nanoparticles as a binding agent, they can For example, bind via a specific antibody to structures of the pathogen and / or the cell, whereby they can be enriched by means of magnetic separation.
  • damming eg by filtration of the sample liquid, can lead to the accumulation of components of pathogens and / or cells.
  • an imaging detection of an exciter and / or a cell can be provided by microscopic magnification and / or image acquisition, in particular by means of a camera, image processing and / or evaluation in a means for image processing and / or a mobile computer unit.
  • a fluorescent signal can be recorded by means of an illumination unit by means of image processing, preferably comprising a magnification unit and a camera, so that fluorescent structures of the pathogen and / or cell can be visualized in the microscopic image.
  • the image processing means and / or the mobile computer unit can, without user intervention, an object recognition of these fluorescent structures and a further evaluation such. an automatic estimation of the exciter numbers per sample volume.
  • the results can also be transmitted to medical professionals by means of the mobile computer unit, which can create a diagnosis and initiate appropriate therapy.
  • the test method can be used as a detection of diseases.
  • diseases are conceivable: infectious diseases, diseases caused by fungi, viruses and / or bacteria, and / or pathogens and / or pathogens.
  • pathogens are also conceivable as pathogens: adenoviruses, amylostomas, ascaris, babesia, bacillus anthracis, Bordetella pertussis, Bordetella parapertussis, Borrelia recurrentis, Brucella sp., Campylobacter sp., Cestoda, Chlamydia psittaci, Clostridium botulinum, Corynebacterium diphtheriae, Coxiella burnetii, human pathogenic Cryptosporidium sp., Ebolavirus, Echinococcus multilocularis, Echinococcus granulosus, Escherichia coli, Enterohe
  • test method is preferably a detection of malaria pathogens conceivable, which occur in particular in a blood sample.
  • test method means for localization, immobilization and / or enrichment for the detection of malaria pathogens can be used in a blood sample. It is possible that at least one structure may be available in infected erythrocytes.
  • the test method may contain a means for permeabilization, in particular saponin and / or ammonium chloride, and for at least one capture ligand, in particular at least one antibody against plasmodial proteins, which preferably can be coupled to at least one magnetic particle, in particular a turbobead, and / or one Labeling ligands, preferably at least one fluorescently labeled antibody against plasmodial proteins and / or at least one DNA dye against plasmodial DNA.
  • a means for permeabilization in particular saponin and / or ammonium chloride
  • at least one capture ligand in particular at least one antibody against plasmodial proteins
  • at least one magnetic particle in particular a turbobead
  • Labeling ligands preferably at least one fluorescently labeled antibody against plasmodial proteins and / or at least one DNA dye against plasmodial DNA.
  • At least one structure of plasmodia and / or the at least one infected erythrocyte in a sandwich structure can be detected and / or at least one infected erythrocyte and / or at least one structure of plasmodia can be detected by means of Damming and / or magnetic separation are caught in a means for localization, immobilization and / or enrichment.
  • the test method can be equipped with a means for localization, immobilization and / or enrichment, which contains at least one microcuvette also in combination with a microfluididic structure into which a sample liquid, preferably a defined amount of whole blood from the fingertip, is taken up.
  • the sample liquid may preferably be provided with lyophilized substances such as an anticoagulant and / or a capture ligand with or without magnetic beads and / or one or more labeling and / or stabilizing agents and / or lysing agents and / or permeabilization are mixed.
  • the mixture can be amplified, for example, by applying a switchable external magnetic field through which the magnetic particles contained can be passed through the solution, herringbone patterns in the channel, vibrations, sound or other mixing methods. Subsequently, the exciters or exciter components bound to the means for immobilization, such as magnetic micro- or nanoparticles, can be immobilized on a viewing window and detected microscopically.
  • the test method may comprise a means for localization, immobilization and / or enrichment with at least one microfluidic structure in combination with a filter structure, such as preferably a sucking filter pad such as present in lateral flow tests.
  • a sample liquid such as a defined amount of whole blood from the fingertip is given.
  • the sample fluid may preferably be mixed with lyophilized substances such as an anticoagulant and / or one or more labeling agents such as antibodies with a fluorescent label or gold particles and / or stabilizers and / or a lysing and / or permeabilizing agent.
  • the liquid can be brought into contact with the filter structure, for example by opening a ventilation opening and / or for example by displacement and / or by buckling of the chip and / or by overcoming a Flußbarriere.
  • the filter structure subsequently sucks up the liquid from the capillary.
  • a capture ligand such as a specific antibody against pathogen antigens can be immobilized on the filter structure.
  • the low Pore size of the filter structure such as a suction pad and the suction of the liquid come the exciters or the components in contact with the capture ligand and are immobilized. Similar to a lateral flow test, this produces a band which, unlike lateral flow tests, does not consist of a protein with bound antibodies but rather of labeled pathogens and / or cells and / or their constituents. Depending on the concentration and labeling method, for example with dye, fluorescent dye, gold particles, beads, this band can be analyzed microscopically and / or photographically and / or by eye.
  • membranes for example, at least one of the following can be used: BA83, BA85, CF1, CF3, CF4, CF5, CF6, CF7, LF1, MF1, VF2, Fusion 5, GF / DVA, AE, FF120HPFFHP.
  • a use for the diagnosis of diseases by means of a test system and / or a test method is conceivable.
  • the use according to the invention brings with it the same advantages as have already been described in detail with respect to the test system according to the invention and the test method according to the invention.
  • diagnostics at the point of care can also be carried out by an untrained person, so that the desired pathogens and / or cells can be detected locally and in real time instead of complex laboratory procedures by the test system.
  • FIG. 1 a shows a schematic representation of a target cell before permeabilization
  • FIG. 1 b schematic representation of a target cell after permeabilization
  • FIG. 2a schematic representation of a reaction vessel as a means for localization, immobilization and / or enrichment
  • FIG. 2b shows a schematic representation of a microchannel as a means for localization
  • FIG. 2c shows a schematic representation of a microchannel with accumulation device
  • FIG. 3 schematic representation of an image acquisition and processing according to FIG.
  • FIG. 1a shows a pathogen and / or an infected cell 10 with specific internal 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 having a compartmentalizing structure 30, such as a cell membrane and / or a cell wall, which substantially separates the cell interior from the cell exterior.
  • target structures 40, 50 for these bacterial species for example, on one or more internal structures 20 such as a thylakoid, ribosome, plasmid, chlorosome, flagellum scaffold, nucleoid, cytoplasmic side of the cell membrane and / or other constituents on which target structures such as antigens can be found.
  • internal structures 20 such as a thylakoid, ribosome, plasmid, chlorosome, flagellum scaffold, nucleoid, cytoplasmic side of the cell membrane and / or other constituents on which target structures such as antigens can be found.
  • a capture ligand 60 added to and / or dissolved in the outer solution, such as a specific antibody, for example with a coupled magnetic nanoparticle 70 such as a turbobead and / or a label ligand 80 such as a labeled-coupled specific antibody 90 such as, for example a fluorophore, quantum dot, enzyme, gold particles and / or other labeling can not penetrate into the interior of the bacterium due to the compartmentalizing structure 30 such as a cell wall and / or lipid membrane and / or other structure.
  • the cell 10 may also be an erythrocyte 10 infected with a malaria pathogen 20 (Plasmodium spec.).
  • the target structures 40, 50 can also be Plasmodium-specific structures that are arranged in the interior of the erythrocyte.
  • the erythrocyte is enveloped by a cell membrane which, as a compartmentalizing structure, essentially separates the cell interior from the cell exterior. It contains specific target structures 40, 50 for these plasmodium species, for example on one or more internal structures 20, such as plasmodial proteins on the cytoskeleton and / or on an inner membrane.
  • Figure 1 b illustrates a cell 10 after addition of a permeabilization agent 10, e.g. Saponin, pore-forming agents, lysing agents, import-triggering agents, hypotonic or hypertonic solutions, and / or other substances.
  • a permeabilization agent e.g. Saponin
  • pore-forming agents e.g. pore-forming agents
  • lysing agents e.g., import-triggering agents
  • hypotonic or hypertonic solutions e.g. Saponin
  • openings 31 as cracks, pores, holes, destabilized membrane areas, e.g. without cholesterol, and / or a receiving process 31 such as transport mechanisms in the compartmentalizing structure 30 arise.
  • scavenger ligand (s) 60 coupled with magnetic nanoparticle 70 and / or labeled ligand 80 added to the outer solution can penetrate into the cell and / or the pathogen and there specifically to the respective target structure 40 and 40 / or 50 bind.
  • Capturer ligand 60 and labeling ligand 80 preferably have no cross-reactivity, i. they bind to different target structures 40, 50, so that preferably a sandwich structure can arise.
  • the label ligand 80 such as a fluorescently labeled antibody
  • a target structure 40 such as a pathogen-typed antigen
  • Figure 2a is a schematic representation of a reaction vessel 121 as a means for localization, immobilization and / or enrichment with contained sample with cells 10, 1 1.
  • the cells 10 may represent target cells and cells 11 thereby non-target cell.
  • the reaction vessel 121 may be, for example, a microreaction vessel such as an Eppendorf reaction vessel and / or a chromatographic column.
  • An associated magnet 130 such as a high gradient magnet and / or a permanent magnet and / or an electromagnet and / or other magnet may generate a magnetic attraction 131 in the reaction vessel 121.
  • this attractive force 131 can act on the magnetic nanoparticles 70, as a result of which these and the target structure 50 bound via the scavenger ligand are attracted and, insofar as possible in the reaction vessel 10, can be enriched and immobilized in the spatial vicinity of the magnet.
  • the bound target structure 50 may be associated with constituents and / or the majority of the cell and / or the exciter 10 so that the bound target structure 50 with attached further constituents is in close proximity to the magnet, such as at the magnet facing Wall of the reaction vessel is enriched.
  • Non-target cells such as uninfected erythrocytes can not be bound by capture ligands and can not be enriched.
  • a labeling ligand such as a fluorescently labeled antibody and / or a fluorescent DNA dye, labeling of structures of the target cell can take place.
  • FIG. 2b shows a schematic representation of a microfluidic structure 140 as a means for localization, immobilization and / or enrichment with contained sample using a capture ligand with magnetic nanoparticle.
  • a microfluidic structure consists of a component with contained capillaries and / or microstructures and / or one or more microchannels.
  • the sample liquid with contained cells 10, 1 1 can flow through the microfluidic structure 140 in the flow direction 141.
  • An associated magnet 130 may generate a magnetic force 131 in a region of the microfluidic structure.
  • This force may act attractively on the magnetic nanoparticle 70, as shown in Figure 2a, and thus act on the coupled capture ligand 60 and a bound target structure 50 and / or preferably a tethered majority of the structure 20 and / or 10.
  • This can lead to an enrichment of the target structure and preferably of the cells 10 and / or the components of the microfluidic structure 140, in particular on the ceiling, the side walls and / or the floor.
  • Cells such as uninfected erythrocytes can not be bound and enriched by capture ligands 60.
  • a targeting of target structure (s) 40 of the target cell 10 can be carried out.
  • FIG. 2 c is a schematic representation of a microfluidic structure 140 as a means for localization, immobilization and / or enrichment with a contained sample and a backflow device in the microfluidic channel.
  • the sample liquid with contained cells 10, 1 1 can flow through the microfluidic structure 140 in the flow direction 141.
  • a backfill device in a region of the microfluidic structure may retain contained cells 10, 11 (target cells and non-target cells) and / or target pathogens 10, while the residual liquid may flow partially into / through the backflow device.
  • labeling of structures of the cell 10 can take place via binding of a labeling ligand 80, such as a fluorescently labeled antibody and / or a fluorescent DNA dye. Marked cells 10 (target cells) among the pent-up cells 10, 11 (target cells and non-target cells) can be detected by means of image processing apparatus.
  • FIG. 3 shows a schematic representation of image acquisition and processing after permeabilization, labeling and / or enrichment in the case of fluorescence labeling and imaging detection.
  • the sample with the contained cells 10 can be irradiated with an excitation light 151, for example light of a wavelength in the range of 300 nm to 950 nm of an excitation illumination 150, such as an LED and / or a laser and / or an illumination source.
  • This excitation illumination may be tuned to stimulate the marker 90 to emit fluorescent light 152, such as light of a wavelength in the range of 300 nm to 1000 nm.
  • Fluorescent light 152 may then be captured by image processing means 160.
  • This image processing means 160 may be associated with a magnification unit 161 and a camera 162.
  • Image data of the camera can be processed and transmitted by means of data transmission 164, preferably wirelessly to a mobile computer unit 163, such as a smartphone.
  • the mobile computing unit 163 may evaluate this image data and also perform user guidance and control of the image processing means.
  • contained cells and / or pathogens can be located and displayed on the images.
  • a diagnosis can be made eg by transmission of the Exciter images to a computer unit 164 as another mobile smartphone by medical professionals done by this.
  • Figure 4 shows a schematic representation of a microfluidic structure 200, e.g. contains a capillary and / or a microchannel and / or other microfluidic structure.
  • a sample receptacle 210 into which a volume of liquid samples is added.
  • This can be, for example, a capillary with a defined volume.
  • the sample receptacle 210 may contain lyophilized substances such as e.g. contain an anticoagulant. The sample can then flow through the microfluidic structure 200, for example due to capillary forces.
  • the sample can be divided there, for example, by means of a branch of a microchannel, so that a first part of the sample can flow through a deposit for a solid substance 220 which has been put there, where it is e.g. lyophilized ligands can dissolve.
  • a mixer / incubation structure the first sample portion may be mixed and incubated with this substance (s).
  • the first sample portion may be merged with the second sample portion and mixed and incubated in a mixer / incubation structure 240.
  • the image processing means may detect the target cells / target agents in the detection area.
  • the flow vorgana may preferably be maintained by a suction device 250 such as a wicking mat and / or a micropump, but may also be by an external device such as a pump.
  • the solution may then be collected in a waste reservoir 260.
  • a microfluidic structure as a test strip may further contain a coding such as a barcode and / or an RFID component for identifying the sample.
  • plasmodia in the ring stage are found predominantly in infected red blood cells (iRBC).
  • the plasmodia contain DNA in contrast to the uninfected red blood cells (RBC).
  • RBC uninfected red blood cells
  • PfEMP-1 protein On the surface of iRBC, various parasitic antigens such as the PfEMP-1 protein are found.
  • PfEMP-1 protein On the surface of iRBC, various parasitic antigens such as the PfEMP-1 protein are found.
  • PfEMP-1 protein On the surface of iRBC, various parasitic antigens such as the PfEMP-1 protein are found.
  • PfEMP-1 protein On the surface of iRBC, various parasitic antigens such as the PfEMP-1 protein are found.
  • PfEMP-1 protein On the surface of iRBC, various parasitic antigens such as the PfEMP-1 protein are found.
  • only one of a pool of many variable, genetically encoded subtypes can be found
  • a parasitic antigen is the ring-infected erythrocyte surface antigen (RESA), which contrary to its name is not found on the surface of the iRBC but on the inside of the membrane of infected erythrocytes on components of the iRBC cytoskeleton, on the parasitophoric membrane and after permeabilization of the Plasmodium membrane also in the cytoplasm
  • RESA ring-infected erythrocyte surface antigen
  • the parasitic DNA can be labeled via a fluorescent DNA dye such as Hoechst 33342.
  • a filter now holds back all cells and cell components.
  • the fluorescent label can be visualized with the magnification unit with image processing unit serving as a fluorescence microscope.
  • a magnetic nanoparticle such as a turbobead PEG streptavidin, to which a biotinylated antibody, such as the monoclonal mouse IgM antibody against Pias.
  • the MNP diffuses into the erythrocytes and binds specifically to structures of the infected RBC.
  • a fluorescent DNA dye and / or a fluorescence-labeled antibody is added, which generates an immunofluorescence of the iRBC and / or plasmodia.
  • the iRBC are immobilized and enriched by means of a magnet and can be detected by fluorescence microscopy be detected by imaging.
  • nanoparticles such as the NanoScreenMag particles of Chemicell GmbH and / or the MyQuVigen nanoparticles of Nvigen Inc. are added as a means of binding and labeling to the permeabilized iRBC.
  • these can be immobilized by means of a magnet and detected by fluorescence microscopy by the optical magnification unit.
  • the image data with the fluorescently labeled iRBC may each be subjected to a first processing by the image processing apparatus such as background correction and / or compression.
  • objects can be searched in the images by means of an object recognition app, whose intensity is above a critical value and which can have a certain size and / or number of pixels and which are thus defined as a pathogen.
  • This evaluation can be done in the image processing device and / or in the mobile computer unit.
  • the object detection can be further secured by appropriate controls such as cell detection with white light illumination and / or fluorescence detection after membrane staining. From the detection of pathogens can be concluded on a diagnosis of the patient.
  • Another possibility as an application example for the detection of malaria pathogens is the addition of saponin as a means for lysis and permeabilization to plasmodium-infected erythrocytes in a whole blood sample.
  • saponin By the action of saponin, the outer erythrocyte membrane is lysed and the parasite can be released from it, while the parasitophorous vacuole is retained.
  • At least one structure such as parasitophoric antigens such as the proteins RAP1 or EXP2 are present on this parasitophoric membrane. These are stored for example in Rhoptrien, Micronemen or Dense Granules and are therefore already present in early trophozoites.
  • At least one catcher ligand 60 in particular at least one antibody to 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 can bind to these.
  • RAP1 or EXP2 such as the monoclonal antibody anti-RAP-1, clone 2.29 or the monoclonal antibody anti-EXP-2, clone 2.2 can bind to these.
  • This antibody is preferably attached to at least one magnetic micro- or nanoparticle 70, in particular a Dynabead or Dynabead muon 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 bound against plasmodial proteins such as RAP1 or EXP2.
  • These Antibodies may 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.
  • At least one (preferably fluorescent) DNA dye can be used against plasmodial DNA in order to detect and / or at least one structure 40, 50, 20 of plasmodia 20 and / or erythrocytes 10 infected with at least one or more plasmodia at least one infected erythrocyte 10 and / or at least one structure 40, 50, 20 of plasmodia by damming and / or magnetic separation in a means for localization, immobilization and / or enrichment 120 such as a microtiter plate with at least 96, in particular 1536 wells, a microcuvette to trap a filter structure without or coated with capture ligands or a fluidic structure.
  • a means for localization, immobilization and / or enrichment 120 such as a microtiter plate with at least 96, in particular 1536 wells, a microcuvette to trap a filter structure without or coated with capture ligands or a fluidic structure.
  • sepsis occurs annually in approximately 18 million patients and has a lethality of approximately 50%. More than 25 different pathogens such as bacteria (meningococci, streptococci, etc.) and / or fungal infections may be responsible for sepsis.
  • pathogens such as bacteria (meningococci, streptococci, etc.) and / or fungal infections
  • bacteria meningococci, streptococci, etc.
  • fungal infections may be responsible for sepsis.
  • streptococci already exist commercial rapid tests. However, these usually consist of several steps, since by means of several reagents first the antigen has to be dissolved out of the cell wall and subsequently detected in an indicator reaction. The high specificity of 95 to 100% is well documented. However, the sensitivity of the tests is, for example, only 70 to 90%; in 10 to 30% the result is false negative.
  • infectious bacteria there is usually a regional and temporal change of surface antigens.
  • a rapid assay according to the invention can be carried out by binding and labeling other antigens which are not accessible to ligands without permeabilization of the cells.
  • other antigens which are not accessible to ligands without permeabilization of the cells.
  • there are various embodiments here such as A) damming of all cells and immunofluorescence detection by means of a fluorescently labeled antibody against an internal, more conserved antigen, B) magnetic capture and parallel fluorescence labeling by means of a fluorescent MNP a specific antibody is coupled and / or C) magnetic capture by means of an MNP, preferably Turbobead, with specific antibody and fluorescent labeling using a second fluorescently labeled antibody and / or a DNA dye.
  • Example of an application of the invention for the analysis of food and / or drinking water detection of Legionella in drinking water
  • Legionella are mobile rod bacteria with an average length of 2 to 5 ⁇ and a diameter of 0.5 to 0.8 ⁇ . They are found in numerous species and serogroups worldwide in surface waters and also in drinking water pipes. Infection with Legionella pneumophila occurs through atomized, atomised water and can lead to the so-called legionnaire's disease, a severe pneumonia, which is fatal in 10-15% of cases. Commercial rapid tests usually include a sampling device, which is sent to a laboratory and analyzed there by means of a bacterial culture on agar plates in about 10 days.
  • one embodiment of the invention may include a test strip designed to filter a volume of drinking water in the range of milliliters to several liters and / or cubic meters such that all contained cells, bacteria and pathogens are retained and placed within the detection range.
  • a test strip designed to filter a volume of drinking water in the range of milliliters to several liters and / or cubic meters such that all contained cells, bacteria and pathogens are retained and placed within the detection range.
  • a microporous filtration membrane having a pore size of 0.45 ⁇ m.
  • a means for permeabilizing the pathogens for example, is introduced from a blister contained in the test strip, so that the filtration membrane is incubated with this.
  • a fluorescently labeled antibody and / or fluorescent nanoparticles is added with antibody that binds specifically to antigens and / or cellular structures of Legionella, which would not be accessible without means for permeabilization and have a locally and temporally largely constant structure.
  • the pathogens can be detected by immunofluorescence by the magnification unit with image processing device imaging.
  • a volume of drinking water in the range of milliliters to several liters and / or cubic meters is first mixed in a mixing chamber with a permeabilization agent and magnetic microparticles such as Dynabeads or nanoparticles such as Turbobeads, to which a specific antibody against Legionella antigens is coupled.
  • Fluorescent staining may be by a labeling ligand such as a fluorescent DNA dye and / or a fluorescently labeled antibody and / or a bound antibody fluorescent nanoparticle.
  • the detection can in turn be done by means of imaging immunofluorescence.
  • a sample in particular a body fluid such as urine and / or stool and / or a food sample and / or drinking water can be used, which can be mixed for dissolution with a buffer. Solid constituents can then be separated off, for example, by filtration, sedimentation and / or centrifugation.
  • Pathogens such as Legionella and / or eggs of the pathogens such as the worms Ascaris, Trichiuris, Amylostoma, Taenia can be lysed and / or permabilized with a means for lysis and / or permeabilization.
  • the pathogens, eggs of the pathogens and / or their constituents can then be labeled with a labeling ligand 80 and / or enriched via a means for localization, immobilization and / or enrichment 120 such as a filter structure and / or magnetic particles and / or by means of imaging Microscopy be detected.
  • a labeling ligand 80 and / or enriched via a means for localization, immobilization and / or enrichment 120 such as a filter structure and / or magnetic particles and / or by means of imaging Microscopy be detected.
  • Microfluidic structure 210 sample intake

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
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Abstract

L'invention concerne un système de test comprenant au moins un des moyens suivants : - un moyen de perméabilisation (110) et/ou de lyse d'au moins un agent pathogène (10) et/ou d'au moins une cellule (10), un moyen de fixation (80) et/ou de marquage (90) de parties (20 et/ou 40 et/ou 50) des agents pathogènes (10) et/ou des cellules (10), - un moyen de localisation, d'immobilisation et/ou d'enrichissement (120) au moins un élément constitutif (40 et/ou 50 et/ou 20 et/ou 10) d'un agent pathogène (10) et/ou d'une cellule (10), - un moyen de traitement d'images (160) comprenant de préférence une unité de grossissement optique (161), permettant ainsi une lecture optique d'au moins un moyen de localisation, d'immobilisation et/ou d'enrichissement (120).
EP17737240.6A 2016-06-30 2017-06-30 Test rapide pour le dépistage d'agents pathogènes et de cellules et procédé correspondant Pending EP3479122A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102016112024.3A DE102016112024A1 (de) 2016-06-30 2016-06-30 Schnelltest für den Erreger- und Zellnachweis und Verfahren
PCT/EP2017/066327 WO2018002327A1 (fr) 2016-06-30 2017-06-30 Test rapide pour le dépistage d'agents pathogènes et de cellules et procédé correspondant

Publications (1)

Publication Number Publication Date
EP3479122A1 true EP3479122A1 (fr) 2019-05-08

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EP17737240.6A Pending EP3479122A1 (fr) 2016-06-30 2017-06-30 Test rapide pour le dépistage d'agents pathogènes et de cellules et procédé correspondant

Country Status (5)

Country Link
US (1) US11796542B2 (fr)
EP (1) EP3479122A1 (fr)
CN (1) CN109564219A (fr)
DE (1) DE102016112024A1 (fr)
WO (1) WO2018002327A1 (fr)

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Publication number Priority date Publication date Assignee Title
DE102019106194B4 (de) * 2019-03-12 2020-12-03 Surflay Nanotec Gmbh Vorrichtung zur spektroskopischen Bestimmung der Bindungskinetik eines Analyten
DE102020117212A1 (de) 2020-06-30 2021-12-30 Marc Becker Speicheltest zur Erkennung von Krankheitserregern
DE102021203409A1 (de) 2021-04-07 2022-10-13 Robert Bosch Gesellschaft mit beschränkter Haftung Verfahren zum Nachweis aktiver Viren
CN113576550B (zh) * 2021-07-15 2024-02-20 温州医科大学附属眼视光医院 一种用于眼部结膜细胞取样的便于调节的结膜细胞刷

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US6636623B2 (en) 2001-08-10 2003-10-21 Visiongate, Inc. Optical projection imaging system and method for automatically detecting cells with molecular marker compartmentalization associated with malignancy and disease
US8105849B2 (en) * 2004-02-27 2012-01-31 Board Of Regents, The University Of Texas System Integration of fluids and reagents into self-contained cartridges containing sensor elements
US20070015179A1 (en) * 2005-04-26 2007-01-18 Trustees Of Boston University Plastic microfluidic chip and methods for isolation of nucleic acids from biological samples
US20100203521A1 (en) * 2007-04-02 2010-08-12 Boston Medical Center Corporation Method for bacterial lysis
DE102007017051A1 (de) * 2007-04-11 2008-10-16 Humboldt-Universität Zu Berlin Dual-markierte Fluoreszenzsonden zur Detektion von Proteinen
US20100279309A1 (en) * 2007-11-19 2010-11-04 Florida Atlantic University Microfluidic chips and systems for analyzing protein expression, and methods of use thereof
US9910036B2 (en) * 2008-05-20 2018-03-06 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
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US9797898B2 (en) * 2008-05-20 2017-10-24 Rapid Pathogen Screening, Inc. Methods and devices for using mucolytic agents including N-acetyl cysteine (NAC)
GB201110454D0 (en) * 2011-06-21 2011-08-03 College The Microfluidic photoporation
EP2746750A1 (fr) * 2012-12-22 2014-06-25 Zendia GmbH Système et procédé de test PoC avec unité informatique mobile
US9957554B1 (en) * 2013-12-19 2018-05-01 National Technology & Engineering Solutions Of Sandia, Llc Microfluidic platform for multiplexed detection in single cells and methods thereof
CN105388288B (zh) * 2015-10-21 2016-08-24 广东和信健康科技有限公司 人呼吸道病原体的流式细胞检测试剂盒、方法和细胞固定液
CN105717310A (zh) * 2016-04-15 2016-06-29 肖乐义 一种检测白细胞中结核分枝杆菌的免疫荧光染色方法及试剂盒

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WO2018002327A1 (fr) 2018-01-04
US11796542B2 (en) 2023-10-24
US20200124602A1 (en) 2020-04-23
CN109564219A (zh) 2019-04-02
DE102016112024A1 (de) 2018-01-04

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