Device and method for analyzing a hormone concentration

Abstract

A microfluidic device (10), in particular a cartridge (100) for diagnosis at the point of care or at home is described, for the parallel, in particular multiplex, determination of a concentration of at least one first analyte of a first analyte type and a concentration of at least one first analyte of a second analyte type in a sample, in particular a urine sample, comprising a first reaction chamber (3a) and at least one second reaction chamber (3b), in particular fluidically connected thereto, wherein at least one specific capture antibody (5a, 5b, 5c) is immobilized in the first reaction chamber (3a) for each analyte of the first analyte type to be detected, and wherein at least one specific capture antibody is immobilized in the second reaction chamber (3b) for each analyte of a second analyte type to be detected, and further comprising a detection reagent, in particular in a reservoir (13) of the microfluidic device (10), which is upstream and comprises specific detection antibodies for binding to the respective analytes and / or comprising competitive analytes for binding to optionally still free specific immobilized capture antibodies (5a, 5b, 5c) in the first (3a) and / or second reaction chamber (3b).

Description

[0001] The present invention relates to a microfluidic device, in particular to a cartridge, for diagnosis at the point of care or at home, to a system comprising the same and to a method for operating the same, according to the preamble of the independent claims. State of the art

[0002] According to theory, the female cycle is divided into menstruation, follicular phase, ovulation, and luteal phase. The relative changes in the relevant variables, namely the hormones, herald the different phases. The concentration ranges of hormones in the blood are known and serve as a basis for gynecologists to diagnose imbalances. Depending on the life stage or life orientation, knowing which cycle phase a woman is in is fundamental for appropriate action, for example, for women who want to have children, as well as for contraception and cycle awareness.

[0003] Basal thermometers and cycle apps, sometimes combined, provide indirect parameters about the status of a woman's cycle. Basal body temperature only rises shortly before ovulation, making it impossible to determine the so-called fertile window (approximately 4 days before). This can result in unwanted pregnancies or a reduced chance of becoming pregnant.

[0004] So-called ovulation tests allow hormone measurements, particularly of luteinizing hormone (LH), which provide a rough indication of the time of ovulation. As with indirect methods, however, the fertile window cannot be fully determined, which can result in the consequences described above. Such ovulation tests are available, for example, as test strips in lateral flow format, whose sensitivity is often low. Furthermore, usually only one analyte is detectable in the sample.

[0005] So-called lab-on-a-chip systems, for example, comprise two main components. The first is a test carrier, for example in the form of a cartridge, which contains structures and mechanisms for manipulating a sample, particularly passive components such as channels or reaction chambers, or active components such as valves, pumps, or mixers. The second main component is a processing unit for controlling the microfluidic processes in the cartridge, such as actuating the valves or pumps, as well as for detecting sample components before, during, and / or after processing.

[0006] WO 2014 / 151456 A2 discloses a multi-chamber measuring system for antibody-based determination of various hormones.

[0007] EP 2 211 891 B1 discloses a parallel measurement and / or determination of peptide hormones and steroid hormones. Disclosure of the invention

[0008] According to the invention, a microfluidic device, in particular a cartridge, for diagnosis at the point of care or at home, a system comprising the microfluidic device, in particular cartridge, and a method for operating the same are provided with the features of the independent patent claims.

[0009] This is based in particular on the fact that the microfluidic device, in particular a cartridge, is configured for the parallel, in particular multiplex, determination of a concentration of at least one first analyte of a first analyte type and a concentration of at least one first analyte of a second analyte type in a sample, in particular a biological sample such as a urine sample. The microfluidic device, in particular a cartridge, comprises a first reaction chamber and at least one second reaction chamber, in particular fluidically connected thereto, wherein at least one specific capture antibody is immobilized in the first reaction chamber for each analyte of the first analyte type to be detected, and wherein at least one specific capture antibody is immobilized in the second reaction chamber for each analyte of the second analyte type to be detected.

[0010] Furthermore, the microfluidic device, in particular a cartridge, comprises an upstream detection reagent comprising specific detection antibodies for binding to the respective analytes and / or comprising competitive analytes for binding to possibly still free specific immobilized capture antibodies in the first and / or second reaction chamber. The detection reagent is stored, for example, in a reservoir upstream of the microfluidic device.

[0011] The advantage here is that a very precise measurement of the concentration of analytes of different analyte types, such as different hormone types, or other different analyte types, whose detection is possible using sandwich and competitive ELISA (enzyme-linked immunosorbent assay), is possible in a sample at the point of care or conveniently at home for diagnosis.

[0012] The respective analytes can be detected specifically and sensitively, so that their concentrations can be determined with the same accuracy as in a laboratory analysis by a doctor or in a central laboratory. The at least one first analyte of the first analyte type is bound by a specific capture antibody immobilized in the first reaction chamber, in particular at a defined location, and the at least one first analyte of the second analyte type is bound by a specific capture antibody immobilized in the second reaction chamber, in particular at a defined location. The binding of the analytes of the at least first and second analyte types to the immobilized capture antibodies occurs in the reaction chambers, in particular in parallel and / or in multiplex, since in each reaction chamber, for example, different analytes of one analyte type can be determined in parallel, each with a specific immobilized capture antibody.

[0013] In comparison to lateral flow-based approaches of the state of the art, the active fluidics of the microfluidic device is used to determine at least the concentration of two, in particular several, analytes of different analyte types in one reaction without requiring a test strip per analyte to be detected, and in particular per analyte type to be detected.

[0014] Compared to a lateral flow format, the microfluidic environment increases sensitivity, specificity, and the degree of multiplexing, while also saving reagents compared to individual tests. Heating, dilution, and washing steps are also possible. Another advantage is that the sample can be prepared for analysis, for example, by dilution.

[0015] The reaction chambers are advantageously fluidically connected to one another and, in particular, constructed analogously to one another. The first reaction chamber is, for example, fluidically connected upstream of the second reaction chamber, meaning that a sample is first fed into the first reaction chamber and then into the second reaction chamber. Alternatively, the sample is divided into two parts in the microfluidic device, and one part is fed into each reaction chamber, so that the analytes bind to the specific, immobilized capture antibodies simultaneously. In a further embodiment, each part of the sample is then fed into the other reaction chamber for binding to the analytes.

[0016] The feature of spatial separation through a separate reaction chamber for each analyte type as well as the possible optimization of the microfluidic steps enables parallelization and simultaneous improvement of sensitivity and specificity.

[0017] Another particularly advantageous feature is that the analyte concentration can be determined at the point of care or at home, making it very simple, convenient, and user-friendly. For this purpose, a urine or saliva sample is used, for example. The advantage here is that it can be obtained painlessly and without anxiety, using a simple, non-invasive method. Alternatively, a blood sample, for example, can also be used.

[0018] Another particularly advantageous feature is that even if, for example, intermediate steps in the detection of the various analyte types are different, they can still be read out using the same device or analyzer, and the analyte concentrations can be determined in parallel and using the same method. This saves time, work steps, containers (especially reservoirs or reagent vials), and the reagents themselves.

[0019] The microfluidic device according to the invention is configured, for example, as a lab-on-chip cartridge, hereinafter referred to as a cartridge. The cartridge includes interfaces to another unit, for example, an analytical device, and may include additional components.

[0020] Further advantageous embodiments of the microfluidic device emerge from the subclaims.

[0021] The reaction chambers have dimensions of 0.5 x 0.5 cm and are made of polycarbonate and / or polypropylene, for example, to avoid reactions of the analytes on uncoated surfaces.

[0022] In an advantageous embodiment, the microfluidic device further comprises an input chamber which is arranged upstream of at least one of the reaction chambers. This means that an input sample is first guided into the input chamber before it reaches the at least one reaction chamber. The input chamber can be heated, for example. In addition, the microfluidic device comprises a microfluidic network made up of various microfluidic channels. A microfluidic channel of the microfluidic network leads from a reservoir to the input chamber. By means of this fluidic connection, for example, a buffer or a reagent can be guided from the reservoir into the input chamber to dilute a sample.

[0023] In a further advantageous embodiment, it is provided that the at least one specific capture antibody immobilized in the first reaction chamber is specific for the luteinizing hormone (LH) and / or the follicle-stimulating hormone (FSH) and / or tyrotropin (TSH) and / or the human chorionic gonadotropin (hCG), and further that the at least one specific capture antibody immobilized in the second reaction chamber is specific for testosterone and / or estrogen and / or progesterone.

[0024] The hormones mentioned are relevant hormones of the female cycle, the benefits of which will be discussed later.

[0025] Particularly advantageously, the first reaction chamber comprises at least immobilized specific capture antibodies for luteinizing hormone (LH), follicle-stimulating hormone and human chorionic gonadotropin (hCG) and the second reaction chamber comprises at least immobilized specific capture antibodies for estrogen and progesterone.

[0026] These hormones reflect a woman's cycle status particularly well, so that they can be used to determine, for example, a woman's entire fertile window very precisely.

[0027] In a further advantageous embodiment, the microfluidic device further comprises an optical and / or electrochemical readout unit for detecting the respective analytes bound to the immobilized capture antibodies. The analyte-specific capture antibodies are located at defined, known positions in the reaction chambers—so-called spots.

[0028] The readout unit is, for example, a camera and / or a luminometer and / or a light source, for example a light-emitting diode (LED) for excitation and / or a photodetector.

[0029] The advantage of using a readout unit compared to lateral flow tests is, for example, that the sensitivity is increased and the time until a result is determined is shortened.

[0030] Furthermore, the microfluidic device comprises an evaluation unit configured to determine a concentration of at least the first analyte of the first analyte type and at least the first analyte of the second analyte type using software comprising an evaluation algorithm, in particular based on artificial intelligence. In particular, the evaluation unit is further configured to compare the determined concentrations of at least the first analyte of the first analyte type and at least the first analyte of the second analyte type with known natural concentrations of the respective analytes in order to use these as a basis for evaluation. In this way, for example, deviations from natural concentrations of the respective analytes can be detected and / or natural or unnatural fluctuations in the analytes can be detected, for example natural or unnatural fluctuations in hormones.The advantage here is that, given sufficient data from a user, the software can, for example, determine deviations from individual hormone levels throughout the cycle. Furthermore, it is advantageous that this provides an overall picture of all specific analyte concentrations, compared to state-of-the-art technology, where this is often only possible selectively for individual analytes.

[0031] Based on this, a recommendation for action and / or information is advantageously issued to the user.

[0032] The advantage is that the use of artificial intelligence enables an evaluation with customer-oriented recommendations for action and / or the output of information, instead of a test strip result that has to be interpreted by the user in a readout window.

[0033] In particular, the microfluidic device further comprises a display unit, for example a display, on which the issued recommended action and / or information and / or the determined analyte concentrations are displayed. Additionally or alternatively, the recommended action and / or the information and / or the determined analyte concentrations are transmitted to an associated app and displayed to the user via this app.

[0034] In an advantageous embodiment, the microfluidic device comprises at least one heating device for controlling the temperature of the input chamber and / or at least one of the reaction chambers or individual areas (spots) thereof. The heating device is, for example, a heater and / or a Peltier element.

[0035] The microfluidic device is, for example, a one-piece measuring device the size of a smartphone, which can be easily transported, for example in a handbag.

[0036] Furthermore, according to the invention, a microfluidic system is provided, comprising a microfluidic device according to the invention, which is designed as a cartridge, and an analysis device, comprising an optical and / or electrochemical readout unit, in particular a camera and / or a luminometer, for detecting the respective analytes bound to the immobilized capture antibodies and an evaluation unit configured to determine the concentration of at least the first analyte of the first analyte type and the concentration of at least the first analyte of the second analyte type by means of software comprising an evaluation algorithm, wherein the analysis device is designed to process the cartridge and / or read out results.

[0037] The above-mentioned statements regarding the readout unit apply here accordingly.

[0038] The evaluation unit is preferably programmed accordingly together with a processor in the analyzer. The further explanations regarding the evaluation unit mentioned above apply here accordingly.

[0039] Furthermore, the analyzer comprises, in particular, a display unit, to which the above-mentioned statements also apply, as well as with regard to the app mentioned therein. Furthermore, the analyzer comprises an optional heating device, to which the above-mentioned statements also apply. The cartridge is designed, for example, as a disposable part, while the analyzer is, for example, a multi-use unit.

[0040] The analyzer is about the size of a smartphone, for example, and can be easily carried in a handbag. The cartridge is smaller than the analyzer, about the size of a matchbox.

[0041] The analysis device (processing unit) and the cartridge can be designed, for example, as described in DE102016222072A1 or DE102016222075A1 and the cartridge can be processed accordingly.

[0042] In a further embodiment of the system, both the microfluidic device, in particular the cartridge, and the analysis device comprise an optical and / or electrochemical readout unit and / or an evaluation unit and / or a heating device. The readout and / or evaluation units can, for example, complement each other.

[0043] The invention further relates to a method for the parallel, in particular multiplex, determination of a concentration of at least one first analyte of a first analyte type and at least one first analyte of a second analyte type in a sample, in particular in a biological sample such as a urine sample, by means of the microfluidic device or by means of the microfluidic system, comprising the following steps: a) Providing the sample and introducing a particularly defined sample volume into the microfluidic device.

[0044] The sample, for example a urine sample, is collected in a vessel and a particularly defined sample volume is introduced into the microfluidic device, in particular a cartridge, by a user, for example by means of a pipette or another aid, for example via a microfluidic inlet or directly into the input chamber.

[0045] The input chamber is temperature-controlled, for example to temporarily stabilize critical samples by cooling or to partially denature them by heat. A particular advantage is that the sample can be taken and analyzed at home or at the point of care, which saves a lot of time.

[0046] Another advantage is that if the sample is urine or saliva, it can be obtained painlessly and without anxiety in a simple, non-invasive way. Alternatively, a blood sample can be used, for example.

[0047] Entering a defined sample volume is particularly advantageous because it allows standardization. d) passing the sample into a first reaction chamber in which at least one specific capture antibody is immobilized at a defined location for each analyte of the first analyte type to be detected, and binding of the at least one first analyte of the first analyte type to this so-called capture antibody.

[0048] The advantage of this approach is that only the appropriate analyte of the first analyte type can bind to the specific capture antibody, thus providing a highly specific and accurate result. Furthermore, the analytes can be determined in multiplex. e) passing the sample, or the part of the sample which was not bound in the first reaction chamber, into a second reaction chamber in which at least one specific capture antibody is immobilized at a defined location for each analyte of the second analyte type to be detected, and binding of the at least one first analyte of the second analyte type to this so-called capture antibody.

[0049] The advantages mentioned in step d) also apply in step e).

[0050] Alternatively, the sample can be divided and passed in parallel into the first and second reaction chambers without passing through the other reaction chamber. Steps d) and e) are thus performed in parallel. Alternatively, the sample can be divided and passed in parallel into the first and second reaction chambers, and then passed into the other reaction chamber, where specific binding of the other analyte type occurs.

[0051] Advantageously, after steps d) and e), a washing step is carried out by adding a washing reagent to the first and second reaction chambers. The washing reagent is advantageously stored upstream, in particular in a reservoir of the microfluidic device, in particular the cartridge. f) adding a specific, in particular upstream, detection reagent into the first and second reaction chamber, in particular from a reservoir of the microfluidic device, wherein the specific detection reagent comprises specific detection antibodies and binds these to the respective analytes already bound to the immobilized capture antibodies and / or wherein the specific detection reagent comprises competitive analytes and binds these to any still free specific immobilized capture antibodies.

[0052] Competitive analytes are artificial analytes to the analytes to be detected.

[0053] All detection antibodies and / or competitive analytes advantageously carry the same label for readout. Optionally, one or more detection antibodies or competitive analytes can also be present without a label or, for example, with a biotin label. In this case, a species-specific secondary antibody is required, for example, that binds the detection antibody and carries the label for readout. In the case of the biotin label, a streptavidin-enzyme complex is required, which binds to the biotin and carries the label for readout. An enzyme, for example, horseradish peroxidase (HRP) or alkaline phosphatase (AP), serves as the label for readout.

[0054] Advantageously, after the addition of the detection reagent, at least one further washing step is performed by adding a washing reagent to the first and second reaction chambers to wash out unbound detection antibodies and / or unbound competitive analytes. The washing reagent is advantageously stored upstream, in particular in a reservoir of the microfluidic device and in particular the cartridge. g) Adding a readout reagent, in particular from a reservoir of the microfluidic device. The readout reagent for detecting the labeled detection antibodies and / or for detecting the labeled competitive analytes is, for example, a substrate such as TMB (3,3',5,5'-tetramethylbenzidine) and causes the label, for example the enzyme of the detection antibody or the competitive analyte, to convert the substrate, thereby causing a color reaction and / or a chemiluminescent reaction in the form of an emission of electromagnetic radiation in the ultraviolet and visible light range, and / or a fluorescent reaction in the form of an emission of electromagnetic radiation.

[0055] For example, a reaction catalyzed by horseradish peroxidase can be read colorimetrically, chemifluorescently, or chemiluminescently. A reaction induced by alkaline phosphates can be read colorimetrically or chemiluminescently.

[0056] A readout unit, in particular a camera, records the intensity of the color reaction and / or the emitted electromagnetic radiation.

[0057] With competitive analytes, if there is a lot of analyte in the sample, few competitive analytes will be bound, because the binding sites of many specific capture antibodies immobilized in the reaction chamber, to which the competitive analytes can then bind, are already occupied by the analyte. Thus, a low signal is detected during readout. If there is little analyte in the sample, the binding sites of many specific capture antibodies immobilized in the reaction chamber remain free, to which the competitive analytes can then bind. A high signal is then detected during readout.

[0058] It is advantageous that for the detection of different analyte types, where in one embodiment at least one analyte type is detectable using labeled detection antibodies and at least one analyte type is detectable using competitive analytes, and thus the intermediate steps are different, the readout step can still be carried out by the same readout system with the addition of the same readout reagent and detectable using the same readout unit. This also enables a high degree of parallelization and multiplexing of analytes.

[0059] Following step g), at least one washing step analogous to the washing steps described above is advantageous. h) Determining the concentration of at least the first analyte of the first analyte type and determining the concentration of at least the first analyte of the second analyte type based on the detected reaction, in particular the color and / or radiation intensity, and in particular comparing this with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations is carried out in the evaluation unit with the aid of an evaluation algorithm, in particular based on artificial intelligence.

[0060] In an advantageous embodiment, the first analyte of the first analyte type is at least one first hormone of a first hormone type, and the at least one first analyte of the second analyte type is at least one first hormone of a second hormone type. It is advantageous if the first hormone type is peptide hormones and the second hormone type is steroid hormones.

[0061] Peptide hormones are each composed of two subunits. The first subunit (alpha subunit) is identical in all peptide hormones, while the second subunit (beta subunit) is specific to each hormone.

[0062] Steroid hormones are derivatives of cholesterol and are very similar in structure.

[0063] It is particularly advantageous that a parallel analysis of different hormone types, particularly peptide and steroid hormones, can be carried out in the first and second reaction chambers. For example, the first hormone type, particularly peptide hormones, is detected in the first reaction chamber, and the second hormone type, particularly steroid hormones, is detected in the second reaction chamber.

[0064] The protocols of the method with the different hormone types, especially peptide and steroid hormones, can be specifically adapted and optimized by separating the detection reactions in the different reaction chambers, for example, with regard to flow rate, reaction temperature in the reaction chambers, mixing or dilution processes, and the concentrations of the detection reagents. This leads to an increase in the sensitivity and specificity of the reactions taking place within a microfluidic device, especially a cartridge.

[0065] In a particularly advantageous embodiment, the at least one peptide hormone is luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) and / or thyrotropin (TSH) and / or human chorionic gonadotropin (hCG), and / or at least one steroid hormone is testosterone and / or estrogen and / or progesterone.

[0066] The advantage of determining the concentrations of these hormones is that they are relevant hormones of the female cycle. Measuring their concentrations allows conclusions to be drawn about these hormones, such as the day of the female cycle or the fertile days. Furthermore, natural or unhealthy hormone concentrations and fluctuations can be determined, which allow conclusions to be drawn about possible illnesses or phases, such as menopause or favorable training phases for athletes.

[0067] In a particularly advantageous embodiment, the concentration of at least the following analytes is determined: luteinizing hormone (LH), follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG), as well as estrogen and progesterone.

[0068] These hormones reflect a woman's cycle status particularly well, so that they can be used to determine, for example, a woman's entire fertile window very precisely.

[0069] In a particularly advantageous embodiment, steps b) and c) are carried out after step a): b) Thermal treatment of the sample to separate at least the first type of analyte contained therein, in particular the peptide hormones, into at least a first and a second subunit.

[0070] For this purpose, the sample is heated, particularly in the input chamber, causing thermal lysis of the peptide hormones into their subunits. This process can be supplemented by an upstream enzyme for the enzymatic cleavage of a subunit.

[0071] The steroid hormones in the sample do not experience any changes or damage due to the thermal treatment. c) Separation of at least the first subunit of the first analyte type, in particular the peptide hormones.

[0072] The advantage here is that sensitivity is increased. The separation of the identically structured first subunits (alpha subunits) of the peptide hormones promotes the specific binding of the detection antibodies to the hormone-specific second subunit (beta subunit) of the peptide hormones, as more hormone-specific epitopes are exposed.

[0073] In a further advantageous embodiment, in steps d) and / or e), the sample is left to rest and / or gently agitated in the respective reaction chamber for a predetermined time, in particular for 1-5 minutes. An incubation period in which the sample remains at rest or gently agitated in the reaction chamber promotes the binding of the analytes to the respective specific immobilized capture antibodies, allowing a very precise determination of the analyte concentration in the sample.

[0074] Furthermore, in a further embodiment, it is advantageous if, in steps d) and / or e), at least a partial temperature control of the first and / or second reaction chamber takes place, in particular at 21-37 °C. The temperature control or heating takes place over the entire chamber or in individual areas (spots), for example in which the specific capture antibodies are immobilized.

[0075] The advantage here is that in this way optimal local binding conditions are available in terms of temperature for the binding of the analytes to the respective specific immobilized capture antibodies.

[0076] In a further advantageous embodiment, steps d) and e) are repeated by passing the sample through a microfluidic circuit. This advantageously increases the chances of the analytes binding to the respective specific immobilized capture antibodies.

[0077] In a further advantageous embodiment, it is provided that in step f) the same specific detection reagent is added to the reaction chambers.

[0078] The detection reagent comprises, for example, specific detection antibodies of all analytes or of all analytes of one analyte type and / or competitive analytes to all analytes or to all analytes of one analyte type and is present, for example, as a mix, in particular upstream.

[0079] This is advantageous because the same detection reagent mix can be used for all analytes, even those of different analyte types, enabling a simple and rapid detection reaction. Furthermore, only one reagent needs to be stored upstream, especially in a reservoir of the microfluidic device, saving space and reagents.

[0080] Alternatively, a separate specific detection reagent is added to each reaction chamber or a specific detection reagent is added to at least one of the reaction chambers for each analyte to be determined, in particular hormone.

[0081] In an advantageous embodiment, after step h), in a step i), based on the respective analyte concentrations, a recommended course of action and / or information, in particular a phase and / or a day in the female cycle and / or a fertile window of a test subject, is calculated and output. The recommended course of action and / or information is displayed in particular on a display unit of the microfluidic device or the analysis device. Alternatively or additionally, the determined concentration of at least the first analyte of the first analyte type and / or of at least the first analyte of the second analyte type can also be output. Additionally or alternatively, the recommended course of action and / or the information and / or the determined analyte concentrations are transmitted to an associated app and output to the user via this app.The advantage of this approach is that, instead of interpreting a strip in the readout window of existing lateral flow tests, the test subject receives a concentration of the analytes and / or information and / or a recommendation for action. This avoids uncertainty on the part of the test subjects, as well as, for example, unwanted pregnancies or unnecessary intake of active substances. Short description of the drawing

[0082] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows: Fig. 1: the schematic representation of a first reaction chamber of a microfluidic device according to the invention in a first embodiment, Fig. 2: the schematic representation of a microfluidic device according to the invention in the first embodiment with a first reaction chamber according to Fig. 1, Fig. 3: the schematic representation of a microfluidic device according to the invention according to Fig. 2, which is designed in the form of a cartridge Fig. 4: the schematic representation of an embodiment of the method according to the invention. Embodiments of the invention

[0083] In Fig. 1 shows a first reaction chamber 3a of a microfluidic device, in particular a cartridge.

[0084] The first reaction chamber 3a comprises, for example, dimensions of 0.5x0.5 cm.

[0085] The material of the chamber side walls is, for example, polycarbonate and / or polypropylene.

[0086] The first reaction chamber 3a comprises a chip 4 with immobilized specific capture antibodies 5a, 5b, 5c for each analyte of a first analyte type to be detected. In the following, this is described by way of example for peptide hormones as the first analyte type. The first antibody 5a binds specifically to a first peptide hormone, for example LH, the second antibody 5b binds specifically to a second peptide hormone, for example FSH, and the third antibody 5c binds specifically to a third peptide hormone, for example hCG. The number three of immobilized specific capture antibodies 5a, 5b, 5c is merely exemplary, so that fewer or more specific capture antibodies can be immobilized, or fewer or more analytes or hormones can be detected. The position of the specific immobilized capture antibodies 5a, 5b, 5c is precisely defined, resulting in a geometric array.

[0087] Furthermore, at least one region of the first chamber 3a can be temperature-controlled. For this purpose, the microfluidic device 10 or the analysis device comprises a Fig. 1, a heating device 7, for example a heater or a Peltier element, is shown only symbolically. If the microfluidic device 10 is designed as a cartridge 100, the cartridge 100 comprises, for example, interfaces to the heating device arranged on the analysis device. At least one side wall of the chamber can be heated, or at least an individual area of ​​at least one side wall, in particular one or more areas (spots) on which at least one immobilized capture antibody is located.

[0088] Furthermore, in Fig. 1 also merely symbolically depicts a readout unit 9 in the form of a camera 9, by means of which the analytes or hormones bound to the specific immobilized capture antibodies 5a, 5b, 5c in the first reaction chamber 3a can subsequently be read out. The readout unit 9 is, for example, a part of the microfluidic device 10 itself or, if the microfluidic device 10 is designed, for example, as a cartridge 100, the cartridge 100 comprises an interface to a readout unit 9 located in an analysis device.

[0089] In Fig. Figure 2 shows a first embodiment of a microfluidic device 10 for point-of-care or home diagnosis, for parallel and multiplexed determination of a concentration of at least one first analyte of a first analyte type and a concentration of at least one first analyte of a second analyte type in a sample. The microfluidic device comprises a first reaction chamber 3a according to Fig. 2 and a second reaction chamber 3b downstream of it. The second reaction chamber 3b is designed analogously to the first reaction chamber 3a, with the difference that at least one specific capture antibody is immobilized in it for each analyte of a second analyte type to be detected. The reaction chambers 3a, 3b are fluidically connected to one another. The first reaction chamber 3a is fluidically connected upstream of the second reaction chamber 3b, i.e., a sample is first fed into the first reaction chamber 3a and then into the second reaction chamber 3b. Alternatively, and not shown in the figures, the sample introduced into the device is divided into two parts, and one part is fed into each reaction chamber 3a, 3b, so that the analytes bind simultaneously to the specific, immobilized capture antibodies.

[0090] The microfluidic device 10 further comprises a not shown upstream detection reagent comprising specific detection antibodies for binding to the respective analytes and / or comprising competitive analytes for binding to possibly still free specific immobilized capture antibodies 5a, 5ab, 5c in the first 3a and / or second reaction chamber 3b. For upstream storage, the microfluidic device 10 comprises Fig. 2 three reservoirs 13, the number of reservoirs 13 being merely exemplary. More or fewer reservoirs 13 may also be present. The reservoirs 13 are fluidically connected to the first reaction chamber 3a and / or to the second reaction chamber 3b, so that a pre-stored sample or reagent can pass from these into at least one of the reaction chambers 3a, 3b. The reservoirs 13 are in Fig. 2 is fluidically connected to an input chamber 1, so that a sample in the input chamber 1 can be diluted by adding a buffer or a reagent from one of the reservoirs 13. The input chamber 1 is located upstream of the first reaction chamber 3a, so that a sample to be analyzed is introduced into the input chamber 1 or is fed into the input chamber 1 via a microfluidic port and a microfluidic channel. The input chamber 1 also comprises a heating device 7.

[0091] Furthermore, the microfluidic device 10 comprises a waste container 15, which is arranged downstream of the second reaction chamber 3b.

[0092] The input chamber 1, the first 3a and the second reaction chamber 3b, the reservoirs 13 and the waste container 15 are connected via a fluidic network of microfluidic channels 19, for example as in Fig. 2, are connected to each other. The microfluidic channels 19 are opened and closed via valves 11. The first 3a and the second reaction chamber 3b each include a fluidic outlet 18.

[0093] Furthermore, in Fig. 2 at least one as in Fig. 1 described readout unit 9 in the form of a camera 9 for detecting the analytes of the first analyte type bound to the immobilized capture antibodies 5a, 5b, 5c in the first reaction chamber 3a and the analytes of the second analyte type bound to the immobilized capture antibodies in the second reaction chamber 3b.

[0094] The readout unit 9 is, for example, a part of the microfluidic device 10 itself or, if the microfluidic device 10 is designed, for example, as a cartridge 100, the cartridge 100 comprises at least one interface to at least one readout unit 9 located in an analysis device.

[0095] One readout unit 9, in particular camera 9, can be provided for each reaction chamber 3a, 3b or one, in particular movable, readout unit 9, in particular camera 9 for reading both reaction chambers 3a, 3b.

[0096] Furthermore, the microfluidic device 10 comprises, for example, a Fig. 2, or, if the microfluidic device 10 is designed, for example, as a cartridge 100, the cartridge 100 comprises at least one interface to at least one evaluation unit located in an analysis device. The evaluation unit is configured to determine a concentration of at least the first analyte of the first analyte type and a concentration of at least the first analyte of the second analyte type.

[0097] In addition, the microfluidic device 10 comprises, for example, a Fig. 2, or, if the microfluidic device 10 is designed, for example, as a cartridge 100, the cartridge 100 comprises at least one interface to at least one display unit located in an analysis device.

[0098] In addition, the microfluidic device, in particular designed as a cartridge, or the analysis device comprises, for example, at least one pump, in particular a membrane pump.

[0099] Thus, the at least one readout unit 9 and / or the evaluation unit and / or the at least one heating device 7 are, for example, part of a microfluidic device 10, which is in the form of a one-piece device. Alternatively, the microfluidic device 10 is according to Fig. 2 as cartridge 100 - as in Fig. 3 - wherein the at least one readout unit 9 and / or the evaluation unit and / or the at least one heating device 7 are not part of the cartridge 1000 and are present in a separate analysis device (not shown). The cartridge then has interfaces to the latter (not shown in the figures). The cartridge 100 can have further components (not shown).

[0100] Fig. 4 shows a flowchart of an embodiment of the method 50 according to the invention for the parallel, in particular multiplex, determination of a concentration of at least one first analyte of a first analyte type and at least one first analyte of a second analyte type in a sample.

[0101] Representing other analyte types, the method according to the invention is described below by way of example for peptide hormones as the first analyte type and steroid hormones as the second analyte type in an embodiment.

[0102] In a step a), for example at home, a defined volume of a biological sample, in particular a urine sample, from a female test subject, for example, is introduced into the microfluidic device 10, in particular the cartridge, for example via a fluidic inlet, for example by means of a pipette.

[0103] The sample is fed into the input chamber 1, which is equipped with a heating device 7. In step b), the sample is thermally treated in the input chamber 1, for example, at 45-65 °C for 1-3 minutes, during which the peptide hormones present in the sample separate into a first and a second subunit. In step c), the first subunit, which has the same structure for all peptide hormones, is separated.

[0104] The separated first subunits—since they are not bound to the immobilized specific capture antibody—are discharged from the first reaction chamber 3a, for example, through the fluidic outlet 18, and are directed, for example, into a waste container (not shown in the figures) of the microfluidic device, in particular the cartridge 10. By separating the first subunit, only the second subunit of the peptide hormones remains in the sample, thus exposing more hormone-specific epitopes.

[0105] Steps b) and c) are optional. If the first subunit of the peptide hormones is not separated, the presence of input chamber 1 is not necessary.

[0106] Subsequently, in a step d), the sample is introduced into the first reaction chamber 3a, in which, for example, four capture antibodies specific for different peptide hormones are immobilized. The first antibody 5a binds, for example, specifically to LH, the second antibody 5b binds, for example, specifically to FSH, the third antibody 5c binds, for example, specifically to TSH, and the fourth antibody binds, for example, specifically to hCG. To support the binding, the sample is allowed to rest and / or gently agitated in the first reaction chamber 3a for a predetermined time, in particular for 1-5 minutes, for example at 21-37°C. In addition, the first reaction chamber 3a is temperature-controlled at least in certain areas, for example in the areas in which the specific immobilized capture antibodies are located.

[0107] Subsequently, in a step e), the sample, with the exception of the peptide hormones bound in the first reaction chamber 3a, is passed into the second reaction chamber 3b, in which, for example, three capture antibodies specific for different steroid hormones are immobilized. The first antibody binds specifically to testosterone, for example, the second antibody binds specifically to estrogen, and the third antibody binds specifically to progesterone, for example. To support the binding of these hormones to the respective immobilized capture antibody, the sample is left to rest and / or gently agitated, as well as at least partially tempered, in the areas in which the specific immobilized capture antibodies are located in the second reaction chamber 3b.

[0108] Steps d) and e) can be repeated, for example, by passing the sample in a fluidic circuit.

[0109] Advantageously, after steps d) and e), a washing step is carried out by adding a washing reagent stored in a reservoir 13 into the first 3a and the second reaction chamber 3b.

[0110] Subsequently, in a step f), a specific detection reagent, in particular one stored upstream in a reservoir 13, is introduced into the first reaction chamber 3a and the second reaction chamber 3b. The specific detection reagent comprises detection antibodies specific for the peptide hormones LH, FSH, TSH, and hCG, which bind to the respective hormones already bound to the immobilized capture antibodies in the first reaction chamber 3a, as well as competitive analytes for testosterone, estrogen, and progesterone, which bind to any specific immobilized capture antibodies still free in the second reaction chamber 3b.

[0111] It is advantageous for all detection antibodies and / or competitive analytes to carry the same label for subsequent readout. An enzyme, such as HRP, serves as the label, which can be read colorimetrically, chemifluorescently, or chemiluminescently.

[0112] Advantageously, after the addition of the detection reagent, at least one further washing step is carried out, which is carried out analogously to the washing step described above.

[0113] Subsequently, in a step g), a readout reagent, in particular from a reservoir 13 of the microfluidic device 10, in particular a cartridge, is fed into the first 3a and the second reaction chamber 3b. The readout reagent is, for example, a substrate such as TMB. The label, for example the enzyme of the detection antibody or the competitive analyte, then converts the substrate, causing a color reaction and / or a chemiluminescent reaction in the form of an emission of electromagnetic radiation and / or a fluorescent reaction in the form of an emission of electromagnetic radiation. A camera 9 serves as the readout unit 9, which records the intensity of the color reaction and / or the emitted electromagnetic radiation.

[0114] Advantageously, after the addition of the reading reagent, at least one further washing step is performed, which is carried out analogously to the washing steps described above. In a subsequent step h), the concentrations of the peptide hormones LH, FSH, TSH, and hCG, as well as the concentrations of the steroid hormones testosterone, estrogen, and progesterone, are determined based on the color and / or radiation intensity detected by the reading unit 9 using software comprising an evaluation algorithm, in particular based on artificial intelligence.

[0115] In addition, the evaluation unit advantageously compares the determined hormone concentrations in the sample with known concentrations, and then evaluates and classifies them. Furthermore, natural or unhealthy hormone concentrations and fluctuations can be determined, which allow conclusions to be drawn about possible diseases or phases, such as menopause or favorable training phases for athletes.

[0116] In a step i), the determined concentrations of the respective hormones are then output and / or, based on the classification and evaluation of the respective peptide and steroid hormone concentrations, a recommendation for action and / or information, in particular a phase and / or a day in the female cycle and / or a fertile window of a test subject, is output, for example via a display unit and / or an associated app. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] WO 2014 / 151456 A2

[0006] EP 2 211 891 B1

[0007] DE 102016222072A1

[0041] DE 102016222075A1

[0041]

Claims

[1] Microfluidic device (10), in particular a cartridge (100) for diagnosis at the point of care or at home, for the parallel, in particular multiplex, determination of a concentration of at least one first analyte of a first analyte type and a concentration of at least one first analyte of a second analyte type in a sample, in particular a urine sample, comprising a first reaction chamber (3a) and at least one second reaction chamber (3b), in particular fluidically connected thereto, wherein at least one specific capture antibody (5a, 5b, 5c) is immobilized in the first reaction chamber (3a) for each analyte of the first analyte type to be detected, and wherein at least one specific capture antibody is immobilized in the second reaction chamber (3b) for each analyte of a second analyte type to be detected, and further comprising, in particular in a reservoir (13) of the microfluidic device (10),upstream detection reagent comprising specific detection antibodies for binding to the respective analytes and / or comprising competitive analytes for binding to possibly still free specific immobilized capture antibodies (5a, 5ab, 5c) in the first (3a) and / or second reaction chamber (3b)., [2] Microfluidic device (10), in particular cartridge (100), according to claim 1, further comprising an input chamber (1), in particular a heatable input chamber, which is arranged upstream of at least one of the reaction chambers (3a, 3b). [3] Microfluidic device (10), in particular cartridge (100), according to one of the preceding claims, wherein the at least one specific capture antibody immobilized in the first reaction chamber (3a) is specific for the luteinizing hormone (LH) and / or the follicle-stimulating hormone (FSH) and / or tyrotropin (TSH) and / or the human chorionic gonadotropin (hCG), and wherein the at least one specific capture antibody immobilized in the second reaction chamber (3b) is specific for testosterone and / or estrogen and / or progesterone. [4] Microfluidic device (10), in particular cartridge (100), according to one of the preceding claims, further comprising at least one optical and / or electrochemical readout unit (9), in particular a camera (9), for detecting the respective analytes bound to the immobilized capture antibodies (5a, 5b, 5c) and an evaluation unit configured to determine a concentration of at least the first analyte of the first analyte type and a concentration of at least the first analyte of the second analyte type by means of an evaluation algorithm, in particular based on artificial intelligence, and in particular further comprising at least one heating device and / or a display unit. [5] Microfluidic system comprising a microfluidic device (10) according to one of the preceding claims, wherein the microfluidic device (10) is designed as a cartridge (100), and an analysis device comprising an electrochemical and / or optical readout unit (9), in particular a camera (9), for detecting the respective analytes bound to the immobilized capture antibodies and an evaluation unit configured to determine the concentration of at least the first analyte of the first analyte type and the concentration of at least the first analyte of the second analyte type by means of an evaluation algorithm, in particular based on artificial intelligence, and in particular further comprising at least one heating device and / or a display unit, wherein the analysis device is designed to process the cartridge (100) and / or to read out results from it. [6] Method (50) for the parallel, in particular multiplex, determination of a concentration of at least one first analyte of a first analyte type and at least one first analyte of a second analyte type in a sample, in particular a urine sample, by means of a microfluidic device (10) according to one of claims 1-4 or by means of a microfluidic system according to claim 5 with the following steps a) Providing the sample and introducing a sample volume into the microfluidic device (10), in particular into the input chamber (1) d) passing the sample into a first reaction chamber (3a) and binding at least a first analyte of the first analyte type to a specific immobilized capture antibody (5a, 5b, 5c) e) passing the sample into a second reaction chamber (3b) and binding at least a first analyte of the second analyte type to a specific immobilized capture antibody f) adding a specific upstream detection reagent into the first (3a) and second reaction chamber (3b), in particular from a reservoir (13) of the fluidic device (10), wherein the specific detection reagent comprises specific detection antibodies and binds these to the respective analytes already bound to the immobilized capture antibodies (5a, 5b, 5c) and / or wherein the specific detection reagent comprises competitive analytes and binds these to any still free specific immobilized capture antibodies, g) adding a readout reagent, in particular upstream in a reservoir (13) of the microfluidic device (10), and detecting a reaction catalyzed by the detection antibodies and / or by the competitive analytes, in particular a color and / or radiation intensity, by means of a readout unit (9), in particular a camera (9). h) Determining the concentration of at least the first analyte of the first analyte type and determining the concentration of at least the first analyte of the second analyte type based on the detected reaction, in particular the color and / or radiation intensity, and in particular comparing this with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations is carried out in the evaluation unit with the aid of an evaluation algorithm, in particular based on artificial intelligence. [7] Method according to claim 6, wherein the at least one first analyte of the first analyte type is at least a first hormone of a first hormone type, in particular a peptide hormone, and wherein the at least one first analyte of the second analyte type is at least a first hormone of a second hormone type, in particular a steroid hormone. [8] The method according to claim 7, wherein the at least one peptide hormone is luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) and / or thyrotropin (TSH) and / or human chorionic gonadotropin (hCG), and / or wherein the at least one steroid hormone is testosterone and / or estrogen and / or progesterone. [9] Method according to one of claims 6-8, wherein after step a) steps b) and c) take place: b) Thermal treatment of the sample to separate at least the first in of this contained analyte type, especially the peptide hormones, in at least a first and a second subunit c) Separation of at least the first subunit of the first analyte type, in particular the peptide hormones. [10] Method according to one of claims 6-9, wherein in steps d) and / or e) the sample is left to rest and / or gently moved in the respective reaction chamber (3a, 3b) for a predetermined time, in particular for 1-5 min. [11] Method according to one of claims 6-10, wherein in steps d) and / or e) at least a partial temperature control of the first (3a) and / or the second reaction chamber (3b) takes place, in particular at 21-37°C. [12] A method according to any one of claims 6-11, wherein steps d) and e) are repeated by circulating the sample. [13] Method according to one of claims 6-12, wherein in step f) the same specific detection reagent is added to the reaction chambers (3a, 3b) or wherein a separate specific detection reagent is added to each reaction chamber (3a, 3b) or wherein a specific detection reagent is added to at least one of the reaction chambers (3a, 3b) for each analyte to be determined, in particular hormone. [14] Method according to one of claims 6-13, wherein after step h) in a step i) the respective concentration of the analytes is output and / or a recommendation for action and / or information, in particular a phase and / or a day in the female cycle and / or a fertile window of a test subject is calculated and output, in particular on a display unit and / or in an associated app.

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