POC TEST SYSTEM WITH MOBILE COMPUTER UNIT AND METHOD

Description

[0001] The present invention relates to a test system including test strips and test method and its use, in particular in the point-of-care (PoC) area, wherein a mobile computing unit comprising an image processing device arranged on an optical magnification unit is used for the analysis of body fluids.

[0002] In research, diagnostics, and numerous other fields of application, analytical laboratory tests used for the qualitative and / or quantitative determination of cells, molecules, ligands, analytes, or their activity or composition, form the basis for far-reaching conclusions and even the development of new methods or devices. These tests are based on the well-established methods of DNA / RNA analysis and protein analysis. Another example is the multitude of analytical procedures and methods employed to measure antibody reactions, also known as immune reactions, which are used to determine (bio)markers and many other substances / analytes. Furthermore, microscopy-based methods in the field of diagnostics have been described.

[0003] Point-of-care (PoC) testing procedures are diagnostic tests that can be performed directly and individually on-site at the patient's / subject's location, rather than in a central laboratory, within a short time.

[0004] For a few parameters, there are point-of-care (PoC) test systems, such as immunochromatographic test strips, which detect a soluble biomolecule, e.g., a hormone or protein, in blood, urine, or saliva via antibody binding and a color reaction. Well-known PoC test strips include pregnancy tests and blood coagulation tests, which are offered with or without a measuring device. Furthermore, related rapid test methods are known, such as the lateral flow test (LFT), flow-through test (FTT), agglutination test (AT), and solid-phase test (SPT). All these methods serve for the rapid detection of analytes and are suitable for visual evaluation in a PoC setting.

[0005] In the PoC area, robust and rapid diagnostics are required that meet the special needs of, for example, emergency medicine, and require high mobility and / or connectivity to medically treating specialists.

[0006] However, no point-of-care (POC) tests exist for a large number of diseases, some of which are life-threatening, that require pathogen or cell diagnostics. These include severe bacterial infections that can lead to sepsis, diseases such as malaria, cholera, and tuberculosis, or the quantification of specific blood cells, such as lymphocytes, in cancer patients. Pathogen and cell analysis still requires complex testing in a diagnostic laboratory, which is correspondingly time-consuming and expensive, and also delays the start of individually tailored treatment or therapy.

[0007] The necessary pathogen and cell diagnostics are illustrated using malaria as an example: In 2010, 216 million cases of malaria were reported from 106 countries. According to the WHO, 655,000 people died from malaria worldwide in 2010 – the majority of them children under 3 years old. This disease is caused by the endoparasite parasite in more than 90% of cases worldwide. Plasmodium falciparum triggered, whereby the disease, if left untreated or insufficiently treated, is fatal in most cases.

[0008] The standard method for diagnosing malaria, according to current technology, is based on microscopy. A small amount of blood is drawn, from which a smear or a preparation, known as a thick drop, is made and stained with Giemsa dye. After fixation and drying of the preparation, the parasite can be identified under the microscope in the red blood cells (erythrocytes) and differentiated by a specialist.

[0009] This method requires not only a trained practitioner, but also a laboratory environment and an expensive microscope. In most malaria-endemic areas, such a well-equipped laboratory is simply not available.

[0010] Furthermore, existing molecular biological tests are expensive and also require in-depth user knowledge.

[0011] Also known are lateral flow rapid tests (supra), which can detect parasite-specific antigens, but not the parasite itself or infected cells, and therefore do not allow pathogen or cell diagnostics.

[0012] Therefore, systems with a powerful mobile computing unit (e.g., a smartphone), as described in the prior art patent DE202010007208U1, are of particular interest. Such test systems are used, for example, to determine blood glucose levels, where a test strip containing patient blood is measured electrochemically, and simple analyses are performed. However, suitable pathogen and cell diagnostics for test systems with a mobile computing unit have not been described. Further test systems are described, for example, by Mudanyali et al. ("Integrated rapid-diagnostic-test reader platform on a cellphone", Lab on a chip, Vol. 12, No. 15, April 16, 2012 (2012-04-16), page 2678) and US 2006 / 222567 A1, which each show smartphone-based systems for different test strips.

[0013] However, there is a strong need to provide a test system with a mobile computing unit that allows for complex diagnostics.

[0014] The complex diagnostics are intended to enable, in particular, pathogen and cell diagnostics from body fluids.

[0015] Therefore, the object of the present invention is to provide a test system that enables effective pathogen and cell diagnostics from body fluids in the PoC area.

[0016] The object of the present invention is achieved by the test system according to claim 1. Further embodiments are defined by the dependent claims.

[0017] In a preferred embodiment, the optical magnification unit is aligned or arranged on the test strip, so that an image or picture, series image or moving image (also called video), preferably at least one contrast image, is produced in the presence of the test strip and the image obtained is captured by the image processing device and read out by the mobile computing unit.

[0018] Furthermore, it is preferred that the planar test strip with a first surface of the optical magnification unit containing at least one objective is arranged perpendicular to the objective axis.

[0019] Within the scope of the present invention, "mobile computing unit" means such a unit, which is provided, for example, by a mobile phone with computing function (commercially available, for example, as a "smartphone") or by a laptop or tablet computer (commonly available commercially as a "tablet" or "tablet computer"), which particularly preferably already includes an integrated image processing device ("iPod ®< , iPhone ®< , Android ®< , iPad ®< , Windows ®< Phone" etc.).

[0020] The mobile computing unit essentially has a central processing unit, also known as a processor, which can perform all the calculation and processing steps required for image processing.

[0021] Furthermore, an image processing device, in the form of a camera, is to be arranged according to the invention for the mobile computing unit. The image processing device is used to capture image data from an optical magnification unit. This can be done by photographing the image produced by the optical magnification unit. This is a particularly advantageous method if the optical magnification unit has optics optimized for a camera. Digital cameras, i.e., those with an image sensor whose data is output to a digital storage medium, are preferred. The image sensor can be a standard CCD sensor. A CMOS sensor can also be used, but is not required. Filters, for example an infrared, a low-pass, and / or a color filter, can advantageously be placed in front of the sensor.

[0022] The digital storage medium can be associated with the image processing device, meaning it can be physically located adjacent to the optics and / or only store image data captured by the image processing device. Therefore, it is conceivable that an image processing device could also be housed in a separate enclosure and transmit data wirelessly or via cable to the mobile computing unit. According to the invention, the image processing device is a digital camera located in a separate unit connected to the smartphone or tablet computer. An integrated solution is also possible, but not supported by the claims, in which a storage medium can be used that also serves other applications. This could be the case, for example, in a smartphone, where storage space is provided for many purposes.

[0023] The image processing device can be operated in a single-image, continuous-image, or moving image (including video) capture mode. In continuous-image mode, several images are captured at fixed intervals after a set resolution, for example, five images at one-second intervals. Such a series of images allows for the easy detection of changes in the subject. If it can be assumed that the subject remains unchanged, comparing the different images can be used to improve image quality.

[0024] The present invention fundamentally provides for image processing. This requires a storage medium for image data and a processor. Current smartphones, as mobile computing units, are equipped with sufficiently powerful processors, often with four processor cores (quad-core), which is desirable but not essential. A clock speed of more than 0.5 GHz is desirable for the processor. This is also achieved by current smartphones or tablet computers (supra).

[0025] For the processing of image data according to the invention, an image processing program is required. It is possible, but usually not necessary, to program all operations to be performed by the program from scratch. Therefore, it is advisable to use comprehensive image processing software that allows adaptation to the requirements of the invention, for example, through freely programmable routines. Smartphones, in particular, offer operating systems that allow the programming and use of small, function-optimized programs ("apps"). Basic program functions (such as data handling) can be implemented in such operating systems with very little programming effort. Therefore, such apps that handle image processing within the scope of the present invention are particularly suitable.

[0026] The image processing may need to be designed for an analysis mode. According to the invention, the aim is not to accurately and sharply represent and / or analyze individual structures of an image, but rather to summarily capture color values ​​or color contrasts, preferably fluorescent colors, in a section of the image. This capture can be qualitative, but is generally also quantitative. For example, a simple quantitative evaluation can determine whether a defined critical value is exceeded by colored areas. The image evaluation can preferably be performed integratively by calculating the sum or integral of specific color values ​​or color contrasts relative to all other values ​​in the image section.The specified color values ​​or color contrasts are expediently a narrow interval of color values, but in individual cases, they can also be a single color value. Such color values ​​can be defined on a conventional color scale, for example, a RAL scale, or the procedure allows the determination of color contrasts or relative color contrasts, possibly in the sense of a ratio of one or more color values. Therefore, within the scope of this invention, the terms color values ​​and color contrasts are used synonymously in the broadest sense.

[0027] The quality of image processing can be improved by sequentially examining individual image sections within a larger field. This can be achieved through simple selection during image processing. Alternatively or additionally, it can also be done by optically magnifying ("zooming") each image section. Alternatively or additionally, it can also be achieved by mechanically moving the sample relative to the optics designed according to the invention, as described above (see below: "dynamic method"). Flowing or transporting the sample or test strip relative to the optics designed according to the invention is particularly preferred. This advantageously allows for the complete acquisition of an inventive sample for image processing.

[0028] Image processing can be advantageously carried out directly during this transport, or image series (also: video) can be recorded during transport and subsequently evaluated.

[0029] As part of the image evaluation, a quality analysis can also be performed. This allows the standard deviation of the results in several image sections to be calculated. Alternatively or additionally, it can be determined how close a calculated value is to a critical value. If the value is very close or if the standard deviation is large, this indicates poor measurement quality. Furthermore, a new test may be requested.

[0030] To utilize the present invention, a program on a computer unit, for example as an app on a smartphone, is therefore useful, in which the following basic functions are combined: reading image data, image processing, and quantitative and / or qualitative output of an image processing and / or analysis result. Additionally, the program can offer a quality analysis related to the quality of the analysis of a sample. Furthermore, linking with additional information is possible (internal or external databases on diseases, addresses of medical services, and the like). The linking of such information can be made dependent on the analysis result.

[0031] The mobile computing unit according to the invention preferably has at least one data connection, preferably also in the form of an internet connection. Using this connection, data can also be stored wholly or partially outside the mobile computing unit (it does not have to be clearly assigned to a single computer and can be stored in external server-based storage, e.g., in a "cloud"). Computing operations can be performed by other computers (in this case, the computing power can also be available via a suitable network and thus not be provided by the selected and permanently designated computer). The data connection, and in particular the internet connection, can be wired or wireless; it can be established, for example, via a WLAN protocol or via a data network according to the UMTS or LTE standard.

[0032] The mobile computing unit should also have input options, such as a keyboard or a touchscreen. A small screen (with a diagonal of approximately five to ten inches) is often sufficient for data output. This screen can also display information on whether the captured image data is of sufficient quality, allowing a new image to be taken immediately if necessary.

[0033] According to the invention, the mobile computing unit is a smartphone or tablet computer and the image processing device is a digital camera located in a separate unit connected to the smartphone or tablet computer.

[0034] Within the scope of this invention, "optical magnification unit" means a unit containing at least one objective or an arrangement of one or more objectives or lenses, as in a microscope, which allows sufficient magnification. This can be achieved by connecting the camera to a commercially available microscope. Alternatively, if such a microscope is used, image data can be transmitted directly to the processing unit. However, within the scope of the present invention, the use of a microscope is generally not mandatory. The camera, for example, of a smartphone, can be combined with a magnifying optic. However, such an option is not covered by the claims. According to the invention, the image processing device is a digital camera located in a separate unit connected to the smartphone or tablet computer.

[0035] According to the invention, the optical magnification unit preferably provides magnifications of at least 10x, 100x, or at least 1000x in the presence of the image processing device, preferably 10x to 500x, 100x to 2000x, and in particular 500x to 1000x, optionally with a resolution of less than 0.5 µm, so that sufficient magnification of cells and pathogens can be achieved. This magnification factor can be fixed or variable.

[0036] The optical magnification unit or image processing device has a fluorescent illumination which is used as excitation light for fluorescent dyes (see below).

[0037] The term "means for receiving a test strip comprising a body fluid" means that a test strip can be arranged and fixed to the optical magnification unit by means of a chamber having a slot or insertion or a holder, in particular in such a way that the image processing device together with at least one optical magnification unit allows an optical read-out of the body fluid.

[0038] Therefore, the invention also relates to a test strip suitable for inclusion in the test system, wherein the test strip is included in a chamber and has only an opening next to a slot or insertion or a holder for the light path of the optical magnification unit.

[0039] In one variant, the chamber can be opened and closed, so that the test strip can be placed or held in place during insertion (hereinafter: static method).

[0040] Preferably, however, a slot or insert is provided on the chamber, allowing the test strip to be inserted and removed by pushing it out (hereinafter: dynamic method). The test strip can therefore be moved or transported relative to the optics according to the invention (supra).

[0041] According to the invention, such a test strip is preferably a planar test strip, preferably consisting of one or more transparent, hard material(s), such as a plastic or glass, capable of absorbing bodily fluid. It is further preferred that the test strip consists of two layers bonded together, such as a thin coverslip and a plastic strip. A synonymous term for test strip could be, for example, "chip".

[0042] The test strip has a sample port, preferably recessed as a cup or funnel, for the application of a defined volume (e.g. 25 µl, 50 µl, 100 µl) of a body fluid sample from a subject, in particular a patient.

[0043] For the purposes of this invention, "body fluid" does not exclusively mean blood, whole blood, urine, saliva (sputum), synovial fluid, cerebrospinal fluid, plasma or serum, or tear fluid, sweat, lymph fluid, intercellular fluid.

[0044] A sample of a patient's / subject's body fluid can be treated with any chemicals, reagents, especially dyes and colorants, particularly fluorescent dyes, possibly including usual excipients and additives.

[0045] Such body fluids contain analytes, especially cells, which may contain infected cells (supra) or pathogens as such.

[0046] "Pathogens" within the meaning of this invention can, for example, but are not limited to, bacteria, fungi, viruses and eukaryotic single-celled organisms such as amoebas or eukaryotic parasites. Furthermore, the term "pathogen" refers to disease-causing agents or pathogens, and not to the following: adenoviruses, Bacillus anthracis, Bordetella pertussis, Bordetella parapertussis, Borrelia recurrentis, Brucella sp., Campylobacter sp., Chlamydia psittaci, Clostridium botulinum, Corynebacterium diphtheriae, Coxiella burnetii, human-pathogenic Cryptosporidium sp., Ebola virus, Escherichia coli, enterohaemorrhagic strains (EHEC), Francisella tularensis, tick-borne encephalitis virus, yellow fever virus, Giardia lamblia, Haemophilus influenzae, hantaviruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, influenza viruses, Lassa virus, Legionella sp., human-pathogenic Leptospira sp.Listeria monocytogenes, Marburg virus, measles virus, mumps virus, Mycobacterium leprae, Mycobacterium tuberculosis / africanum, Mycobacterium bovis, Neisseria meningitidis, Norwalk-like virus, poliovirus, Pseudomonas aeruginosa, rabies virus, Rickettsia prowazekii, rotavirus, rubella virus, Salmonella paratyphi, Salmonella typhi, Shigella sp., Trichinella spiralis, varicella-zoster virus, Vibrio cholerae O1 and O139, Yersinia enterocolitica, Yersinia pestis, Treponema pallidum, HIV, Echinococcus sp., Plasmodium sp., Toxoplasma gondii. The invention also includes multi-resistant pathogenic bacteria (MRSA), such as Streptococcus pneumoniae, Streptococcus aureus, etc.

[0047] The term "analytes" does not exhaustively include substances, chemical compounds such as dissolved macromolecules (viruses, nucleic acids, protein complexes, proteins, peptides) or small molecules (organic molecules, hormones, vitamins, metabolites, drugs), etc.

[0048] The term "patient" encompasses any subject, regardless of symptoms or disease, and includes both humans and animals, especially mammals.

[0049] The sample port can be supplied with (dried) chemicals, agents, auxiliaries (e.g. EDTA, heparin for anticoagulation), in particular dyes for histological cell staining or, most preferably, agents for lysis of infected cells (e.g. infected erythrocytes after infection with Plasmodium), optionally with release of the pathogens (e.g. by means of ammonium chloride, saponin, sucrose, etc.), which are preferably dissolved by the introduced sample of a body fluid (hereinafter: sample).

[0050] In a preferred embodiment, the sample flows through the test strip, particularly preferably via at least one incorporated or applied (micro)channel with a rectangular, trapezoidal, or arcuate cross-section. The channel can be a capillary or allow flow by gravity. Preferably, the (micro)channel consists of an optically transparent material such as glass or a plastic, preferably a fluoropolymer with a refractive index similar to that of water, to enable optical detection.

[0051] Therefore, the invention also relates to a test strip containing a sample port and at least one (micro)channel, in particular at least one capillary.

[0052] In a preferred embodiment, the sample port includes a first funnel-shaped depression (10) into which one or more drops of capillary blood are placed. This first funnel is subdivided in its lower region into several, preferably three, subordinate compartments (20, 30, 40), for example by means of suitable dividers (50). The arranged dividers (50) define the size of the compartments (20, 30, 40) and consequently the partial volume attributable to each compartment.

[0053] Therefore, the invention relates to a test strip containing a sample port, wherein a first funnel-shaped depression (10) is divided into several compartments (20, 30, 40). The compartments (20, 30, 40) each have an opening, preferably including a cell filter ("blood filter"), which opens into a common connecting channel (90), wherein, however, the compartments (20, 30, 40) have different path lengths to the detection chamber (110) and are arranged one after the other (see Figure 4 At least one compartment (20) contains a pre-prepared, preferably dried, staining solution, optionally including a binder and other excipients, in particular agents for lysing infected cells (e.g., infected erythrocytes after infection with Plasmodium). The largest compartment (30) is preferably positioned centrally to compartments (20) and (40) and allows the introduction of a defined quantity of body fluid into compartments (20) and (40) (see Figure 4 ).

[0054] These cell filters ("blood filters") allow larger blood cells to be retained, but pathogens to pass through.

[0055] The fluid containing the pathogens from (40) passes through the connecting channel (90) into the (micro)channel or detection chamber (110) due to capillary forces (capillary suction).

[0056] As a result, an air bubble forms at the junction (60) of the supply line from (40) at the connecting channel (90). This air bubble separates the liquids from (40) and (20) and stops the flow from (20) until the reservoir (40) is empty. Subsequently, the liquid level in (20) can push the air bubble into the channel coming from (40), so that the staining solution flows into the connecting channel (90) to the subsequent (micro)channel or detection chamber (110) with a time delay.

[0057] Therefore, the invention relates to a test strip containing a sample port, wherein a first funnel-shaped depression (10) is divided into several compartments (20, 30, 40), and a sample, after separation, is fed unmixed and sequentially into a (micro)channel via a common connecting channel (90).

[0058] This advantageously allows for a defined aliquoting of the sample, including the use of staining solutions and auxiliary materials. The guaranteed reproducibility of the test results is particularly beneficial.

[0059] Therefore, the invention relates to a test strip containing a sample port, wherein a first funnel-shaped depression (10) is divided into several compartments (20, 30, 40), wherein the compartments (20, 30, 40) have openings and a sample enters the (micro)channel or detection space (110) from one compartment via a common connecting channel (90) in a first step and in a second step the (bound) pathogens are stained from a dye in a second compartment.

[0060] The Figures 3 to 5 show a preferred embodiment of a microstructured sample port (sample intake) for one or more drops of body fluid, such as capillary blood, as described above.

[0061] The test strip can have one or more fields in which specific binders (130), such as antibodies, ligands, proteins or aptamers, are preferably bound in the form of a monolayer to a first surface of the test strip.

[0062] These fields, along with the binder, advantageously allow for multiplex analyses, i.e., of different cells, infected cells, and pathogens side by side.

[0063] Such binders are accessible to professionals and are commercially available, for example.

[0064] For example, for Streptococcus pneumoniae, isolated C-polysaccharide or the pathogen's surface protein PspA can serve as a target for a specific binder. The gene for the surface protein PspA is publicly available under NCBA Gene ID 932894. A protein can be produced from this gene sequence using standard procedures, which can then be used as a target for isolating a binder from the species mentioned above. RNA binders can be isolated using the SELEX method (systematic evolution of ligands by exponential enrichment). SELEX is a method for isolating ligands with high affinity for a target protein from a pool of varying DNA or RNA sequences (molecular library).

[0065] The oligonucleotide sequences obtained using SELEX are called aptamers (review: Ellington AD, Szostak JW, Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures, in Nature, 355 (1992), 850-852).

[0066] For this purpose, DNA molecules with variable sequences, i.e., a large population of different DNA variants, are generated on a DNA synthesizer. These randomized DNA sequences are flanked by defined DNA segments that are recognized by predefined PCR primers. One of these defined segments contains the signal sequence for initiating transcription by T7 RNA polymerase. Typically, approximately 10¹⁵ to 10⁴⁵ different DNA sequences are generated for one SELEX run.

[0067] Incubation between the nucleotide sequences and the targets can take place in the immobilized state or in solution. Washing steps separate the sequences that are only weakly bound or not bound at all to the targets from the bound sequences.

[0068] The improved binders are then amplified using reverse transcription (only for RNA) and polymerase chain reaction (PCR). This results in a pool of DNA or RNA molecules with improved binding properties to the ligand compared to the original pool of molecules. This step completes the cycle, which is then typically repeated eight to twelve times until a desired aptamer is obtained.

[0069] The sequence of the aptamer thus found can be determined using standard methods and enables the production of the aptamer in large quantities.

[0070] Commercial antibodies against PspA are available from various manufacturers, for example goat anti-S. pneumoniae antibodies bN-18 or bF-19 from Santa Cruz Biotechnology Inc.

[0071] The specific binder can therefore be, for example, a nucleic acid aptamer, a PNA sequence, a monoclonal antibody, an antibody fragment, or an affibody (Gebauer M, Skerra A: Engineered protein scaffolds as next-generation antibody therapeutics. In: Curr Opin Chem Biol. June 2009).

[0072] Of course, the invention can be applied analogously to other pathogens, as illustrated below: For example, in the case of Staphylococcus aureus (bacterium), isolated surface antigen B, enterotoxin B, or the manganese transporter protein MntC of the pathogen can serve as a target for a specific binder. Commercial antibodies are also available, for example, the monoclonal mouse anti-S. aureus antibodies clones S622 and S643 from Santa Cruz Biotechnology Inc. Similarly, in the case of Pseudomonas aeruginosa (bacterium), isolated mucoid exopolysaccharide of the pathogen can serve as a target for a specific binder. Commercial antibodies against serotypes 9 and 10 of P. aeruginosa are also available, for example, mouse anti-P. aeruginosa antibody clone 276 / 11 or 95 / 159 from Santa Cruz Biotechnology Inc., for example in the case of Haemophilus influenzae (bacterium), can serve as a target for a specific binder pneumococcal histidine triad D (PhtD), the pneumolysin toxoid (dPly) or the NTHi protein D (PD) of the pathogen.Commercial antibodies are also available, e.g., monoclonal mouse anti-H. influenzae antibodies from antibodies-online Inc. For example, in the case of Mycobacterium tuberculosis (bacterium), a specific binder surface antigen, PE_PGRS16, of the pathogen can serve as a target. Commercial antibodies against M. tuberculosis are also available, e.g., polyclonal rabbit anti-M. tuberculosis antibodies from abcam plc. For example, in the case of Neisseria gonorrhoeae (bacterium), a specific binder surface antigen, Omp85 (GenBank: AAC17600.1), of the pathogen can serve as a target. Commercial antibodies against N. gonorrhoeae are also available, e.g., polyclonal rabbit anti-N. gonorrhoeae antibodies from antibodies-online Inc., for example in the case of Corynebacterium diphtheriae (bacterium), can serve as a target for a specific binder surface antigen protein, DIP1281 or SpA, of the pathogen. Commercial antibodies against C.Antibodies against diphtheriae are also available, e.g., monoclonal mouse anti-C. diphtheriae antibodies from antibodies-online Inc. For example, in the case of Bordetella pertussis (bacterium), selected aptamers of the pathogen can serve as a target for a specific binder LOS-A or for specific adhesins necessary for attachment to the ciliated epithelium. Commercial antibodies against B. pertussis are also available, e.g., monoclonal mouse anti-B. pertussis antibodies from antibodies-online GmbH. For example, in the case of Salmonella enterica (serovar Typhi) (bacterium), nonfimbrial protein SiiE of the pathogen can serve as a target for a specific binder. Commercial antibodies against S. enterica are also available, e.g., monoclonal mouse anti-S. enterica antibodies from antibodies-online Inc.In the case of Yersinia pestis (bacterium), a specific binder such as Adhesin Ail, Plasminogen Activator (Pla), or pH 6 antigen (Psa) of the pathogen can serve as a target. Commercial antibodies against Y. pestis are also available, e.g., monoclonal mouse anti-Y. pestis antibodies from abcam plc. For example, in the case of Campylobacter jejuni (bacterium), the FlhF protein of the pathogen can serve as a target. Commercial antibodies against C. jejuni are also available, e.g., monoclonal mouse anti-C. jejuni antibodies from abcam plc. For example, in the case of Legionella pneumophila (bacterium), the PAL protein or other components of the bacterial lipopolysaccharide layer of the pathogen can serve as a target. Commercial antibodies against L. pneumophila are also available, e.g., monoclonal mouse anti-L. pneumophila antibodies from antibodies-online Inc. ZBIn the case of Listeria monocytogenes (bacterium), autolysin, a specific binder of the pathogen, can serve as a target. Commercial antibodies against L. monocytogenes are also available, e.g., polyclonal rabbit anti-L. monocytogenes antibodies from antibodies-online Inc. Similarly, in the case of hepatitis B virus, PreS1 (a specific envelope protein) can serve as a target. Commercial antibodies against HBV are also available, e.g., monoclonal mouse anti-HBV antibodies from antibodies-online Inc. Finally, in the case of hepatitis C virus, the E1-E2 dimer of the viral envelope can serve as a target. Commercial antibodies against HCV are also available, e.g., monoclonal mouse anti-HCV antibodies from antibodies-online Inc. For example, in the case of Japanese Encephalitis Virus, the binder domain II of the envelope protein (E) of the virion of the pathogen can serve as a target.Commercial antibodies against Japanese encephalitis virus are also available, e.g., monoclonal mouse anti-Japanese encephalitis virus antibodies from antibodies-online Inc. For example, in the case of Candida albicans (fungus), a specific binder polysaccharide of the pathogen's cell wall can serve as a target. Commercial antibodies against C. albicans are also available, e.g., monoclonal mouse anti-C. albicans antibodies from antibodies-online Inc. For example, in the case of Aspergillus fumigatus (fungus), a specific binder protein Cfr1 of the pathogen can serve as a target. Commercial antibodies against A. fumigatus are also available, e.g., monoclonal mouse anti-A. fumigatus antibodies from antibodies-online Inc. For example, in the case of Plasmodium falciparum (bacterium), a specific binder protein of the pathogen, such as MSP-1 (merozoite surface protein), AMA1 (apical membrane antigen 1), or EMP1 (also: PfEMP1 / knob), can serve as a target.Commercial antibodies against P. falciparum are also available, e.g., monoclonal mouse anti-P. falciparum antibodies from Santa Cruz Biotechnology Inc. For example, in the case of Plasmodium vivax (bacterium), a specific binder, the circumsporozoite (CSP) protein of the pathogen, can serve as a target. Commercial antibodies against P. vivax are also available, e.g., monoclonal mouse anti-P. vivax antibodies from Santa Cruz Biotechnology Inc.

[0073] The person skilled in the art is therefore able to provide suitable binders, preferably polyclonal or monoclonal antibodies, aptamers, for the respective cells, infected cells or pathogens.

[0074] Therefore, the invention relates to a test system according to claim 1. Further embodiments are defined by the dependent claims.

[0075] Such binders can, for example, be printed, fixed or immobilized and serve as catchers for the cells, infected cells, pathogens, which are consequently concentrated from the sample (liquid) on the surface of the test strip.

[0076] As previously explained, the test strip preferably has several fields with identical or different binders (captures). Different fields can be assigned different binders (captures), each of which specifically concentrates cells, infected cells, or pathogens.

[0077] The (micro)channel in question preferably runs through the fields, allowing the body fluid sample to come into contact with the binders on the fields.

[0078] To prevent a laminar flow of the cells on the test strip or through the said channel and to increase the probability of contact with the binders or binder-coated fields, disruptive structures for turbulence can be placed in the channel or on the test strip, which are preferably round, oval, triangular, square, rectangular, or polygonal protrusions positioned perpendicular to the flow direction.

[0079] Furthermore, the test strip can contain a (nano)porous membrane (140) (pore size 50 to 400 nm) that generates additional outflow and consequently draws the cells, infected cells, or pathogens (160) onto the test strip or fields toward the binders (captures) (130). To generate sufficient capillary flow, the test strip can include a capillary pump (170). Alternatively, filter paper, nonwoven fabric, or the like (120) can also be used on the underside or adjacent to the strip. Preferred embodiments are described in the Figures 7 and 8 explained.

[0080] Therefore, the invention relates to a foreground test strip containing a (nano)porous membrane (140) (pore size 50 to 400 nm), and optionally a capillary pump (170) or filter paper, nonwoven fabric (120).

[0081] In a further embodiment, the (micro)channel can be equipped with an electric field, the field preferably being oriented at a right angle to the surface of the test strip. Consequently, charged exciters can mostly be directed towards the binder (130). The electric field can be applied along the test strip and / or the (micro)channel using conventional electrodes and a sufficient field strength.

[0082] The binders recognize the targeted cells, infected cells or pathogens using specific targets, such as surface markers, capture them from the sample flow and bind, fix or immobilize them to a first surface of the test strip or designated field.

[0083] In a particular embodiment of the test strip according to the invention, bars or markings (300) can be placed between the fields, preferably orthogonal to the flow direction (see Figure 6a ) or a DotCode (310) may be printed on it (see Figure 6b They are advantageously used for the visual delimitation of the fields for image processing and for determining the speed at which the test strip is passed by the user using evaluation software (see also "dynamic method").

[0084] A barcode (320) containing standard test strip information, such as the type of test (malaria pathogen, HIV, or batch number) and / or the test strip's expiry date, can optionally be printed before or after (downstream or upstream) the fields.

[0085] In another embodiment, a mimetic can also be used that is on the order of size of a cell or pathogen and has the same or different binders and is also specifically bound by the binders on at least one field.

[0086] Such a mimetic is, for example, a (latex or polystyrene) bead, magnetic sphere or spheroid containing the same or different binders, which have been added to the sample or placed in at least one field on the test strip.

[0087] This mimetic allows for the quantitative detection of any analyte from body fluids, such as dissolved macromolecules (viruses, nucleic acids, protein complexes, proteins, peptides) or small molecules (organic molecules, hormones, vitamins, metabolites, drugs). For this purpose, an "artificial cell" (mimetic) must be presented to which these soluble molecules or viruses specifically bind or are addressed, thus creating a situation that corresponds to the presence of pathogen-specific markers on real cells.

[0088] Therefore, the invention also relates to a test system according to the invention comprising a test strip having one or more fields having at least one identical or different binder suitable for binding one or more analytes, cells, infected cells, pathogens, optionally by means of a mimetic, in particular in the form of beads, magnetic spheres and / or spheroids.

[0089] According to the invention, the field can be of any size and any designation. However, the designation of several fields that are spatially separated from one another is preferred.

[0090] This leads to an accumulation of infected cells or pathogens in at least one of these areas, while the liquid sample continues to flow, preferably into a collection reservoir (e.g., sponge, reservoir, fleece, fabric, etc.) due to gravity or capillary action. Alternatively, a terminal micropump can also be used (e.g., micropump mp6, Bartels Microtechnik GmbH). The cells or pathogens can retain their physiological shape in the liquid.

[0091] The accumulation of infected cells or pathogens can also be achieved mechanically through a sieve or filter, or by narrowing the aforementioned (micro)channel.

[0092] Cells can also be biologically enriched, for example, as specific aggregates. Such aggregates can be obtained, for instance, by adding fibrinogen to erythrocyte rolls.

[0093] Therefore, in a further embodiment, the invention relates to a test system comprising a test strip, wherein at least one or more analytes, cells or pathogens are localized, in particular mechanically localized.

[0094] In a second step, the infected cells or pathogens can be stained by adding a staining solution to the now-emptied sample port, or from a blister pack that is opened mechanically and opens into the channel. Depending on the analyte and test configuration, the staining is preferably a simple histological stain of the cytoplasm and DNA in the cell nuclei, or an immunochemical stain (e.g., Giemsa).

[0095] In this process, additional antibodies, peptides, or aptamers (hereinafter: staining ligands) are preferably used, which bind to a second, independent binding site on the surface of the immobilized or trapped cells. These ligands are preferably coupled to a fluorescent dye, so that their binding to the cell surface generates a sufficient fluorescence signal, which is made visible against the background by optical magnification, thus producing individual imageable color values ​​or color contrasts, as described above.

[0096] Therefore, the invention also relates to a test system wherein the analytes, cells, pathogens bound on the test strip are color-marked by means of a dye ligand coupled to a dye, preferably a fluorescent dye, and are consequently accessible to an image evaluation according to the invention.

[0097] This dual recognition in the "sandwich system" by the binder (capture ligand) on the one hand and by the (subsequent) staining ligand on the other hand allows for unambiguous typing of the infected cells or pathogens and excludes false positives through non-specific binding of other cells.

[0098] Appropriate binders (capture ligands) and staining ligands for cells and pathogens are described in the literature and can be used accordingly by experts. For example, infected cells (malaria, cholera, etc.) exhibit corresponding surface markers that can be recognized by the binders (capture ligands and staining ligands).

[0099] The method enables the enrichment and unambiguous visual identification of cell types such as malaria-infected erythrocytes, CD4+ cells or also infected cells in HIV diagnostics, as well as the identification and typing of bacteria in the diagnosis of e.g. sepsis, cholera, tuberculosis, etc.

[0100] Enrichment and recognition are even possible in multiplexing for a wide variety of cell types or pathogens. For this purpose, different fields within the (micro)channel are coated with different binders, each specifically capturing and enriching a desired cell type or pathogen. Immunochemical labeling is achieved by flowing a mixture of preferably fluorescently labeled ligands for all desired cell types or pathogens, which are thereby specifically stained.

[0101] The background signal of the specific marker, caused by the staining solution remaining in the channel, is not relevant for the measurement, as the staining solution should preferably be low in concentration. For example: With a volume of 0.1 µl in the microchannel (capillary) (0.1 mm x 0.02 mm x 50 mm) and 0.0002 µl over a field (0.1 mm x 0.1 mm x 0.2 mm), and 50 µl of low-concentration staining solution used, the solution is exchanged 500 times in the capillary and 250,000 times over the field.

[0102] The test strip therefore allows, after sample addition in the first flow step, the specific enrichment and immobilization of certain cells or pathogens from the body fluid sample. By the subsequent addition of a staining solution, all remaining unbound cells are washed from the microchannel, and the cells or pathogens are preferably specifically labeled with a (fluorescent) dye. A particular advantage is that, according to the invention, no additional washing step is required.

[0103] To further enhance the color signal, polymers, preferably dendrimers, can be used which carry an intensely luminous fluorophore at each of their up to 1,000 ends (also called "amplifier complex").

[0104] In addition to fluorophores, these dendrimers are equipped with other ligands such as antibodies, peptides, and aptamers. The resulting molecules thus resemble the aforementioned staining ligands, but with a much higher number of fluorophores per molecule. Furthermore, different dendrimers can be used, each containing different fluorophores that can be specifically excited at the same or different wavelengths and fluoresce with maxima at different wavelengths.

[0105] Once the staining liquid has completely distributed itself in the canal, or has flowed through it, unbound staining ligands, possibly reinforced with dendrimers, are evenly distributed in the canal.

[0106] Cells immobilized in the channels carry marker-bound staining ligands on their surfaces. The concentration of staining ligands in the staining solution is preferably selected such that staining ligands are concentrated on the infected cells (pathogens) compared to the free staining ligands present in the solution.

[0107] As a result, infected cells appear in contrast as lighter colored / fluorescent dots or areas against a colored / fluorescent background, while uninfected cells remain invisible.

[0108] Surprisingly, according to the invention, only 2-5 detected pathogens are sufficient, and assuming a capture efficiency of 10% in 10 µl of whole blood, this equates to 20-50 pathogens. The detection limit is therefore particularly advantageous at 3-5 pathogens per µl of blood. Commercially available test strips (lateral flow tests) that detect malaria via the indirect detection of soluble pathogen proteins in the blood (HRP II, aldolase, S-antigen) have a sensitivity of approximately 100 pathogens per µl.

[0109] For comparison: In malaria, a parasitemia of 2-5 pathogens per µl is found in the symptomatically rather inconspicuous initial stage, whereas during a fever attack, parasitemias of 100,000 pathogens per µl can occur.

[0110] In the presence of (excitation) illumination, the image evaluation according to the invention of the color contrasts of lighter colored / fluorescent cells against a colored / fluorescent background can be performed. By identifying lighter spots or areas, the number of infected cells per volume of sample can be determined by counting and calculation (integration, supra), since the sample volume and the binding efficiency of the fields are known. Calibration may be necessary.

[0111] Due to the above measures, when the sample-loaded test strip is inserted into the test system, sufficient color intensity and color contrast, along with sufficient color values, can be obtained for one or more images, which can be read out in the mobile computer unit via the optical magnification unit and image processing device according to the invention, as described above, so that effective image processing can take place.

[0112] In the case of the aforementioned "static method," images or series of images (including video) with sufficient diagnostic information can be obtained, enabling qualitative analysis or diagnosis. For example, the result might be that a malaria parasite is present in the body fluid.

[0113] However, the aforementioned "dynamic method" is particularly preferred, whereby multiple fields of the test strip can be optically read in a series of images or video sequences. The test strip should preferably be transported / moved uniformly relative to the optical magnification unit.

[0114] This can be done simply by carefully pulling out the test strip manually, or in particular by means of an electric motor-driven transport, a spring-loaded winding motor or a gas spring that is compressed by insertion.

[0115] Therefore, the invention also relates to a method defined by claim 12.

[0116] Furthermore, it is preferred that the test strip is transported or moved relative to the optics according to the invention, in particular to the optical magnification (so-called "dynamic method").

[0117] Therefore, the invention relates to an additional optional process step b'.), wherein the test strip is transported or moved relative to the optics according to the invention, in particular to the optical magnification.

[0118] The optical magnification unit, including means for receiving a test strip containing a body fluid, can be the subject of an attachment for the mobile computing unit, such as a smartphone, or in the form of a docking station (via connector / interface plug or wirelessly) or Bluetooth® or WLAN device.

[0119] The attachment, in the case of a smartphone or tablet, will typically cover at least part of the back. It can be mechanically, magnetically, or detachably connected to the smartphone or tablet. An attachment can also be offered in the form of a desktop stand (for example, a "docking station"). The desktop stand may have its own (rechargeable) power supply and also perform additional functions, such as serving as a charging station for the smartphone or tablet. A desktop stand or other attachment can be sold independently of a smartphone or tablet and, together with a standard smartphone or tablet, becomes a powerful analysis station.

[0120] The test system can be designed accordingly by the aforementioned features, even if these features relate to the method according to the invention.

[0121] Therefore, the invention relates to an attachment or table stand, in particular in the embodiment of a docking station, comprising an optical magnifier and means for receiving a test strip as described above, which is / can be connected to a mobile computing unit containing an image processing device. Furthermore, the invention relates to the use of a test system or test method according to the invention for diagnosing diseases of all kinds, as defined by claim 13, in particular infectious diseases, wherein, by means of evaluation and identification according to the invention of cells, infected cells, pathogens, and significant analytes, a conclusion can be drawn about a disease in a patient / test subject.This applies in particular to diseases caused by fungi, viruses and bacteria, or pathogens or agents, especially those such as adenoviruses, Bacillus anthracis, Bordetella pertussis, Bordetella parapertussis, Borrelia recurrentis, Brucella sp., Campylobacter sp., Chlamydia psittaci, Clostridium botulinum, Corynebacterium diphtheriae, Coxiella burnetii, human-pathogenic Cryptosporidium sp., Ebola virus, Escherichia coli, enterohaemorrhagic strains (EHEC), Francisella tularensis, tick-borne encephalitis virus, yellow fever virus, Giardia lamblia, Haemophilus influenzae, hantaviruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, influenza viruses, Lassa virus, Legionella sp., and human-pathogenic Leptospira sp.Listeria monocytogenes, Marburg virus, measles virus, mumps virus, Mycobacterium leprae, Mycobacterium tuberculosis / africanum, Mycobacterium bovis, Neisseria meningitidis, Norwalk-like virus, poliovirus, Pseudomonas aeruginosa, rabies virus, Rickettsia prowazekii, rotavirus, rubella virus, Salmonella paratyphi, Salmonella typhi, Shigella sp., Trichinella spiralis, varicella-zoster virus, Vibrio cholerae O1 and O139, Yersinia enterocolitica, Yersinia pestis, Treponema pallidum, HIV, Echinococcus sp., Plasmodium sp., Toxoplasma gondii. The invention also includes multi-resistant pathogenic bacteria (MRSA), such as Streptococcus pneumoniae and Streptococcus aureus. Examples and figures:

[0122] These examples serve solely to illustrate the invention, without limiting the invention to these examples. Description of the characters:

[0123] Figure 1 describes a preferred embodiment of a test strip according to the invention. Figure 2 shows the insertion of a test strip into the device according to the invention using a smartphone (below), wherein an optical magnification including a suitable chamber is arranged above the digital camera. Figure 3 : Top view showing the funnel-shaped sample port (10), but the sample port (10) is subdivided into three sub-funnels (compartments: 20, 30, 40) by means of partitions (50). Figure 4 : Supervision of the arrangement with the (compartments: 20, 30, 40) and connecting channel (90) including the incoming microchannels. Figure 5 : Cross-section of the arrangement to the Figures 3 and 4 including version (70) and edition (100). Figure 6a: Top view of a test strip (70) having a nanoporous membrane (140) with applied fields including binders (captures) (130), wherein a sample containing pathogens (160) can enter the detection chamber (110) through the connecting channel (90) and has markings (300). Furthermore, a barcode (320) is provided. Figure 6b : Top view of a test strip (70) having a nanoporous membrane (140) with applied fields including binders (captures) (130), wherein a sample containing pathogens (160) can enter the detection chamber (110) through the connecting channel (90) and has a DotCode (310). Furthermore, a barcode (320) is provided. Figure 7Cross-section of a test strip (70) including support (100) comprising a nanoporous membrane (140) and additionally underlying filter paper (120) and applied fields with binders (captures) (130), wherein a sample containing pathogens (160) can enter the detection chamber (110) through the connecting channel (90). The arrows illustrate the suction direction achieved for the pathogens (160) due to the resulting outflow. Figure 8 Cross-section of a test strip (70) including support (100) comprising a nanoporous membrane (140) and adjacent filter paper (120) or capillary pump (170) and applied fields including binders (captures) (130), wherein a sample containing pathogens (160) can enter the detection chamber (110) through the connecting channel (90). The arrows illustrate the direction of suction of the pathogens (160) through the resulting outflow. Figure 9On a nanoporous membrane (140), fields containing binders (130) are applied, consisting of a layer (200) and scavengers, in this case: antibodies (180). The layer (200), e.g., made of polyethylene glycol (PEG), prevents non-specific binding of cells / pathogens to the surface. PEG forms a gel-like layer, thus increasing antibody contact (180) with cells / pathogens through penetration and greater spatial flexibility of the antibodies (180).

[0124] The pathogens (160) glide over the surface and bind to the capture receptors (180) with their surface antigens (190). Only the underside of the pathogens (160) is blocked. Other, differing antigens (210) on the upper side of the pathogens are freely accessible and bind to added detection antibodies (220). Conventionally, these are fluorescently labeled by direct coupling to a small number of fluorophores.

[0125] Preferably, a signal enhancer complex is used that binds up to hundreds of fluorophores to an antibody and generates a stronger fluorescence signal. The detection antibody (220) is coupled to a dendrimer (250) via a linker (240), e.g., a single PEG molecule. This dendrimer contains hundreds of chemical groups, e.g., amino groups, on its surface, to each of which a fluorophore has been coupled.

Claims

1. A test system for use in the diagnosis of diseases, comprising i.) a mobile computing unit, specifically a smartphone or a tablet computer, ii.) an image-processing device together with at least one optical magnification unit, and iii.) means for receiving a test strip (70), wherein a bodily fluid containing cells, infected cells, pathogens (160), is applied to the test strip, and iv.) the test strip (70) containing one or more capture ligands (130) which bind cells, infected cells, pathogens from iii.), wherein these cells, pathogens, are marked in color by means of a stain ligand (220), which is conjugated with a fluorescent dye (250), characterized in that the test strip is arranged such that the image-processing device together with at least one optical magnification unit allows for optical read-out of the bodily fluid, and the image-processing device is a digital camera, which is located in a separate unit connected to the smartphone or the tablet computer, wherein the optical magnification unit or image-processing device comprises one or more light sources of any kind, and at least one fluorescent light, wherein the test strip contains a sample port having a first funnel-shaped depression (10), which is subdivided into a plurality of compartments (20, 30, 40), wherein at least one compartment (20) contains a provided staining solution together with the stain ligand, selected from the group of antibodies, peptides, and aptamers, which are conjugated with the fluorescent dye.

2. The test system according to any one of the preceding claims, wherein the optical magnification unit contains at least one objective or an arrangement of one or more objectives or lenses, in particular in the form of a microscope.

3. The test system according to any one of the preceding claims, wherein the optical magnification unit, in the presence of the image-processing device, reaches magnifications of at least 10 times or at least 100 times or at least 1000 times.

4. The test system according to any one of the preceding claims, wherein means for receiving a test strip comprise a chamber containing a slot or insert or a holder for a test strip.

5. The test system according to any one of the preceding claims, containing a test strip, wherein the test strip is pulled in and out via a slot or insert by means of electromotively driven transport, a spring wind-up motor, or a gas spring.

6. The test system according to claim 1, comprising a test strip containing at least one (micro)channel.

7. The test system according to any one of the preceding claims, containing a test strip that has one or more fields that have at least the same or different capture ligands, in particular mimetics, beads, magnetic balls, and spheroids.

8. The test system according to any one of the preceding claims, containing a test strip, characterized in that the test strip contains the sample port and at least one (micro)channel.

9. The test system according to claim 8, containing a test strip, characterized in that the test strip comprises at least one capillary, along with markings (300), a DotCode (310), and / or a bar code (320).

10. The test system according to any one of the preceding claims, containing a test strip, characterized in that the test strip contains a porous membrane (140), a capillary pump (170), or filter paper or mat (120).

11. The test system according to any one of the preceding claims, containing a test strip, characterized in that the bodily fluid is selected from blood or whole blood.

12. A test method for being carried out on a test system according to any one of the preceding claims, comprising the following steps: a.) a bodily fluid is applied to a test strip, wherein at least one capture ligand binds a pathogen in a localized manner on the test strip, b.) a fluorescent dye is applied to the test strip, wherein a pathogen from a.) is marked in color, c.) the test strip is transported or moved relative to the optical magnification, d.) at least one contrast picture from b.) is available for image processing and is evaluated in the mobile computing unit.

13. The use of a test system or test method according to any one of the preceding claims for diagnosing diseases.

14. The use of a test system or test method according to claim 13 in the point-of-care sector.