Device and method for analyzing an analyte concentration

Abstract

The invention relates to a microfluidic device (10), in particular a cartridge (100) for a diagnosis at the point of care or at home, for parallel, in particular multiplex, determination of a concentration of at least two analytes (4a, 4b, 4c), in particular of different analyte types, in a sample, in particular a urine sample, the device comprising a first reaction chamber which has a first filter (14a), and at least one second reaction chamber which is fluidically connected to the first reaction chamber and has a second filter (14b), wherein at least one first specific capture antibody (5a) for a first analyte (4a) to be detected is immobilized on the first filter (14a), and wherein at least one second specific capture antibody (5b) for a second analyte (4b) to be detected is immobilized on the second filter (14b), and further comprising specific detection antibodies (3a, 3b, 3c), located upstream in the microfluidic device (10), for binding to the respective analytes (4a, 4b, 4c), and / or competitive analytes, located upstream in the microfluidic device (10), for binding to the at least one specific immobilized capture antibody (5a, 5ab) on the first filter (14a) and / or on the at least one second filter (14b).

Description

[0001] Description

[0002] title

[0003] Device and method for analyzing analyte concentration

[0004] The present invention relates to a microfluidic device, in particular a cartridge, for point-of-care or home diagnosis, to a system comprising the latter, and to a method for operating the same, according to the preamble of the independent claims.

[0005] State of the art

[0006] The female cycle is theoretically divided into menstruation, the follicular phase, ovulation, and the luteal phase. The relative changes in the involved factors, namely the hormones, signal the start of these different phases. The concentration ranges of these hormones in the blood are known and serve as a basis for gynecologists to diagnose imbalances.

[0007] Depending on the phase of life or lifestyle, knowing which phase of the cycle a woman is in is fundamental for taking the appropriate action, for example for women who want to have children, as well as for contraception and cycle awareness.

[0008] Basal thermometers and cycle-tracking apps, sometimes used in combination, provide indirect parameters for a woman's cycle status. Basal body temperature only rises shortly before ovulation, meaning the so-called fertile window (approximately 4-5 days before ovulation to 1 day after) cannot be fully determined. This can result in unintended pregnancies or a reduced chance of becoming pregnant.

[0009] So-called ovulation tests allow for hormone measurement, particularly of luteinizing hormone (LH), which provides a rough indication of the time of ovulation. However, as with indirect methods, the fertile window cannot be fully determined, which can lead to the consequences already described. Such ovulation tests are available, for example, as lateral flow test strips, which often have low sensitivity. Furthermore, usually only one analyte is detectable in the sample.

[0010] Systems such as Quanovate's Mira or Inito's Fertility Monitor demonstrate that the simple lateral flow format can be further developed into a detection format for 2-3 analytes in a multiplex. However, sample preparation is not possible with such formats, and the sensitivity is low.

[0011] So-called lab-on-a-chip systems comprise, for example, two main components. The first is a test carrier, for example in the form of a cartridge, which includes structures and mechanisms for manipulating a captured sample, in particular 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 components of the sample before, during, and / or after processing.

[0012] In WO 02 / 071060 A2, various methods for the specific detection of a hormone using antibodies are described.

[0013] US patent 2021 / 0138071 A1 discloses a number of different possible antibodies for the specific binding of corresponding antigens of hormones to be investigated, along with detection methods.

[0014] Disclosure of the invention

[0015] 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 a cartridge, and a method for operating the same, with the features of the independent claims, are provided.

[0016] This is based in particular on the fact that the microfluidic device, especially the cartridge, is configured for the parallel, and in particular multiplex, determination of the concentration of at least two analytes, in particular of different analyte types, in a sample. The sample is, for example, a biological sample, such as a urine sample. The microfluidic device comprises a first reaction chamber with a first filter and at least one second reaction chamber fluidically connected to it with a second filter, wherein at least one first specific capture antibody of a first analyte to be detected is immobilized on the first filter, and wherein at least one second specific capture antibody of a second analyte to be detected is immobilized on the second filter, such that the filters are designed to be "analyte-specific".The filter membranes have macroscopic structures, meaning that they are designed to allow passage through the various analytes and other sample components.

[0017] The microfluidic device can have any number of additional reaction chambers with further filters, each containing a further specific capture antibody of another analyte to be detected. In this way, the concentration of any number of analytes can be determined multiplexically by sequentially connecting filters within the fluidic network of the microfluidic device.

[0018] Furthermore, the microfluidic device includes upstream specific detection antibodies for binding to the respective analytes and / or upstream competitive analytes for binding to specific immobilized capture antibodies on the first and / or second and / or subsequent filters.

[0019] The advantage here is that a very precise measurement of the concentration of analytes, especially of different types of analytes, such as different types of hormones, or other different types of analytes, which can be detected 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.

[0020] 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 at the doctor's office or in the central laboratory.

[0021] The at least two analytes are bound by specific capture antibodies immobilized on the first and at least one second filter. A key advantage is that the binding of the analytes to the immobilized capture antibodies on the filters occurs in parallel and / or in multiplexing, since, for example, one analyte with a specific immobilized capture antibody can be determined simultaneously on each filter. Compared to a lateral flow format, the microfluidic environment increases sensitivity, specificity, and the degree of multiplexing, and also reduces reagent consumption compared to individual assays. Furthermore, this allows for heating of the reaction chambers as well as staining and washing steps.

[0022] It is also advantageous that the sample can be prepared for analysis, for example by dilution.

[0023] The reaction chambers with the filters are fluidically connected and, in particular, have an analogous design. For example, the first reaction chamber is fluidically connected upstream of the second, meaning that a sample is circulated in a fluidic loop, first entering the first reaction chamber and then the second. Alternatively, the sample is divided into two parts within the microfluidic device, and each part is introduced into a separate reaction chamber, allowing for simultaneous binding of the analytes to the specific, immobilized capture antibodies. Subsequently, each part of the sample is passed into the other reaction chamber for analyte binding.The feature of spatial separation through a separate reaction chamber for each analyte type, as well as the optimization of the microfluidic steps possible there, enables parallelization and simultaneous improvement of sensitivity and specificity.

[0024] Furthermore, the sequential and parallel execution of different detection formats (e.g., sandwich and competitive ELISA) is facilitated. A particularly advantageous feature is that analyte concentrations 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 this can be obtained painlessly and without anxiety, using a simple, non-invasive method. Alternatively, a blood sample can also be used.

[0025] A further advantage is that even if, for example, intermediate steps in the detection of different analyte types differ, they can still be read out using the same device or analyzer, and the analyte concentrations can be determined in parallel and via the same method. This saves time, work steps, containers (especially reservoirs or reagent vials), and the reagents themselves.

[0026] The microfluidic device according to the invention is, for example, designed as a lab-on-a-chip cartridge, hereinafter referred to as the cartridge. The cartridge includes interfaces to another unit, for example an analyzer, and may include further components.

[0027] Further advantageous embodiments of the microfluidic device are set forth in the dependent claims.

[0028] The reaction chambers, for example, have dimensions of 0.5 x 0.5 cm and are made of polycarbonate and / or polypropylene to avoid reactions of the analytes on uncoated surfaces.

[0029] The filter membranes consist of materials such as silicon dioxide (SiO2) or aluminum oxide (Al2O3). Meshes made of copper, nickel, gold, molybdenum, titanium, stainless steel and / or aluminum can also be used.

[0030] In one embodiment, it is advantageous if at least one of the filters is electrically conductive and the reaction chamber containing it includes two electrodes, so that an enzymatic reaction in the reaction chamber can be electrically detected. The electrodes, for example made of platinum or gold, are deposited onto the filter in a ring-shaped manner or are rod-shaped and attached to the filter. The filter itself can also be made of a conductive material such as gold or platinum. In this case, for example, a second electrode is attached to the upper side of the reaction chamber.

[0031] The electrical measurement begins with the addition of a readout reagent and the application of a voltage between the electrodes. If an analyte or a competitive analyte is bound to the filter membrane, an enzymatic redox reaction starts near the membrane, which is detected by the two-electrode system. A voltage (depending on the enzyme system used) is applied between the two electrodes, and the resulting current is measured; its magnitude is proportional to the analyte concentration. If no analyte is present, there is no current increase during the reaction.

[0032] An advantage of electrical detection is that active electrochemical readout can occur during the flow through the microfluidic device (live measurement), for example, with circulating pumps. Readout can also be performed in a static state. This provides advantageous degrees of freedom with respect to the readout process. Furthermore, in an alternative or additional embodiment, it is advantageous if at least one of the filters includes an integrated dye, allowing for colorimetric detection of an enzymatic reaction in the reaction chamber. In this case, the enzyme reaction leads, for example, directly to a color change of the dye integrated into the filter, which can then be read out colorimetrically. During the readout, the concentration of the bound analytes is measured based on the absorption of light in a specific wavelength range.In this case, the dye is, for example, non-covalently bound to the filter with low affinity. In the presence of a suitable enzyme, the affinity increases and the dye is converted. Alternatively, for example, the dye is released in a voltage-dependent manner when a suitable voltage is applied.

[0033] Alternatively, a pH-sensitive dye is integrated into the filter, allowing the enzyme reaction to be indirectly detected colorimetrically via a color change of the filter due to a change in pH caused by the enzyme reaction. The pH-sensitive dye is, for example, a fluorescein derivative or an absorbing substance that changes color depending on the pH value, such as phenol red.

[0034] A key advantage of colorimetric detection is that no expensive metals need to be applied to the filters, which would then have to be disposed of after processing. Furthermore, only a camera, which can consist of simple light-emitting diodes (LEDs), is required for readout.

[0035] In an advantageous embodiment, the microfluidic device further comprises an input chamber, which is located upstream of at least one of the reaction chambers. This means that an input sample is first directed into the input chamber before entering a reaction chamber. The input chamber can be heated or temperature-controlled, for example, to allow critical samples to be stabilized temporarily by cooling or to at least partially denatured by heat. In one embodiment, specific detection antibodies against the target analytes, labeled for identification, are stored upstream of the input chamber. These detection antibodies can be stored in the input chamber in either dry or liquid form.

[0036] In a further advantageous embodiment, it is provided that the specific capture antibodies immobilized on the filters are specific for luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) and / or tyrotropin (TSH) and / or human chorionic gonadotropin (hCG), and / or testosterone and / or estrogen and / or progesterone.

[0037] The hormones mentioned are relevant to the female cycle. Information about their concentrations in a sample can provide insights into many aspects of the female cycle.

[0038] Particularly advantageous is the fact that the first filter in the first reaction chamber contains immobilized specific capture antibodies for luteinizing hormone (LH), the second filter in the second reaction chamber contains immobilized specific capture antibodies for follicle-stimulating hormone (FSH), and further filters in additional reaction chambers contain immobilized specific capture antibodies for human chorionic gonadotropin (hCG), estrogen, and progesterone. These hormones provide an exceptionally accurate representation of a woman's menstrual cycle status, allowing for the precise determination of, for example, a subject's entire fertile window.

[0039] In a further advantageous embodiment, the microfluidic device further comprises at least one electrical and / or optical readout unit for detecting the respective analytes bound to the immobilized capture antibodies.

[0040] The readout unit is, for example, a current / voltage source and / or a camera, which may consist of or include simple LEDs. One advantage of using a readout unit compared to lateral flow tests is the increased sensitivity.

[0041] Furthermore, the microfluidic device includes an evaluation unit set up to determine the concentration of at least two analytes using software comprising an evaluation algorithm, in particular based on artificial intelligence.

[0042] In particular, the evaluation unit is further configured to compare the determined concentrations of at least two analytes with known natural concentrations of the respective analytes in order to use this as a basis for evaluation. This allows, for example, the detection of deviations from natural concentrations of the respective analytes and / or the identification of natural or unnatural fluctuations of the analytes, such as natural or unnatural fluctuations of hormones. A key advantage is that, given sufficient data from a user, the software can, for example, determine deviations from an individual's hormone levels throughout their menstrual cycle. Furthermore, it is beneficial that this provides a comprehensive overview of all determined analyte concentrations.

[0043] Advantageously, based on this, a recommendation for action and / or information is issued to the user.

[0044] 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 needs to be interpreted by the user in a reading window.

[0045] In particular, the microfluidic device further comprises a display unit, for example a screen, on which the issued action recommendation and / or information and / or the determined analyte concentrations are displayed. Additionally or alternatively, the action recommendation and / or the information and / or the determined analyte concentrations are transmitted to a corresponding app and displayed to the user via this app.

[0046] In an advantageous embodiment, the microfluidic device comprises at least one heating device for temperature control of the input chamber and / or at least one of the reaction chambers. The heating device is, for example, a heater and / or a Peltier element.

[0047] The microfluidic device is, for example, a one-piece measuring device the size of a smartphone, which can be conveniently transported, for example in a handbag.

[0048] Furthermore, according to the invention, a microfluidic system is provided, comprising a microfluidic device according to the invention, which is configured as a cartridge, and an analyzer comprising an electrical and / or optical readout unit, in particular a current and / or voltage source and / or a camera, for detecting the respective analytes bound to the immobilized capture antibodies, and an evaluation unit configured to determine the concentration of the at least two analytes by means of software comprising an evaluation algorithm, wherein the analyzer is configured to process the cartridge and / or read out results from it. The above-mentioned descriptions regarding the readout unit apply accordingly. The evaluation unit is advantageously programmed together with a processor in the analyzer. The further above-mentioned descriptions regarding the evaluation unit apply accordingly.

[0049] Furthermore, the analyzer includes, in particular, a display unit, to which the above-mentioned statements also apply, as well as to the app mentioned therein. The analyzer also includes an optional heating unit, to which the aforementioned statements also apply.

[0050] The cartridge, for example, is designed as a disposable item, while the analyzer, for example, is a multi-use unit.

[0051] The analyzer is about the size of a smartphone, making it easy to carry, for example, in a handbag. The cartridge, in comparison, is smaller, about the size of a matchbox.

[0052] The analyzer (processing unit) and the cartridge can be designed, for example, as described in DE102016222072A1 or DE102016222075A1, and the cartridge can be processed accordingly.

[0053] In another embodiment of the system, both the microfluidic device, in particular the cartridge, and the analyzer comprise an electrical and / or optical readout unit and / or an evaluation unit and / or a heating device. The readout and / or evaluation units can, for example, complement each other.

[0054] The invention further relates to a method for the parallel, in particular multiplex, determination of a concentration of at least two analytes in a sample, in particular in a biological sample such as a urine sample, using the microfluidic device or the microfluidic system, comprising the following steps: a) providing the sample and introducing a particularly defined sample volume into the microfluidic device, in particular into the input chamber.

[0055] The sample, for example a urine sample, is collected in a container and a defined sample volume is introduced into the microfluidic device, in particular a cartridge, by a user, for example via a microfluidic inlet, for example into an input chamber, using a pipette or other aid.

[0056] The input chamber can be optionally temperature-controlled to temporarily stabilize critical samples through cooling or to partially denature them through heat. A particular advantage is that the sample can be taken and analyzed at home or at the point of care, saving considerable time and effort.

[0057] Another advantage is that if the sample is a urine or saliva sample, it can be obtained painlessly and without anxiety, using a simple, non-invasive method. Alternatively, a blood sample can be used, for example.

[0058] Inputting a defined sample volume is particularly advantageous, as this allows for standardization. b) Passing the sample into a first reaction chamber with a first filter and into at least a second reaction chamber with a second filter, binding the first analyte to a first specific capture antibody immobilized on the first filter, and binding the at least one second analyte to a second specific capture antibody immobilized on the second filter.

[0059] The advantage of this method is that only the appropriate analyte can bind to the specific capture antibodies, thus obtaining a highly specific and accurate result. Furthermore, the analytes can be determined using multiplex assays. The binding of the analytes to the respective filters can be demonstrated, for example, using an ELISA – depending on the analyte to be detected, for instance, a sandwich ELISA or a competitive ELISA. In both cases, a readout reagent is added for the detection of labeled detection antibodies and / or for the detection of labeled competitive analytes. The readout reagent causes the label, for example, the enzyme of the detection antibody or the competitive analyte, to react with a substrate, resulting in, for example, an electrochemical reaction in the form of a redox reaction and / or a color reaction.c) Determining the concentration of the first filter-bound analyte and of at least one second filter-bound analyte, and in particular comparing these with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations is carried out in an evaluation unit using an evaluation algorithm, in particular based on artificial intelligence.

[0060] In one embodiment, the analyte concentration in step c) is determined electrically based on an enzymatic reaction, in this case a redox reaction, detected by a two-electrode system in the respective reaction chamber. The respective filters are electrically conductive, and the reaction chamber comprises two electrodes. Before the readout, a readout reagent (enzyme substrate) and, optionally, mediator molecules, which serve as electron shuttles to the membrane during the enzymatic redox reaction, are flushed through the microfluidic device and, for example, circulated. Both the readout reagent and any required mediator molecules are located upstream of the microfluidic device, for example, in a reservoir.

[0061] The electrical measurement begins with the addition of the readout reagent. If an analyte is bound in the sandwich or a competitive analyte is present at the membrane, the enzymatic redox reaction starts near the membrane, which is detected by the two-electrode system. A voltage must be applied between the two electrodes, and the resulting current is recorded. The current response is proportional to the analyte concentration. An advantage of this system is that active electrochemical readout is possible while the readout reagent flows through the microfluidic device. Furthermore, no camera is required for readout; only a simple circuit board is needed to apply a voltage and record the current, resulting in a more cost-effective system.

[0062] In an alternative or additional embodiment, the concentration of the analytes in step c) is determined colorimetrically by means of a color change of a filter, in particular a pH-sensitive filter.

[0063] In this case, the enzyme reaction leads, for example, directly - or in the case of a pH-sensitive filter indirectly via a change in pH value caused by the enzyme reaction - to a color change of the dye integrated into the filter, so that the color change can be advantageously read out colorimetrically close to the filter.

[0064] In one embodiment, it is advantageous if at least one of the analytes is a peptide hormone, in particular luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) and / or tyrotropin (TSH) and / or human chorionic gonadotropin (hCG), and if at least one of the analytes is a steroid hormone, in particular testosterone and / or estrogen and / or progesterone.

[0065] A particular advantage is that the microfluidic device allows for the parallel analysis of different hormone types, especially peptide and steroid hormones. For example, a peptide hormone is detected in the first reaction chamber on the first filter, particularly via a sandwich ELISA, and a steroid hormone is detected in the second reaction chamber on the second filter, particularly via a competitive ELISA. While the intermediate steps differ, the readout step can be performed using the same readout system with the same readout reagent and detectable by the same readout unit. This also enables a high degree of parallelization and multiplexing of analytes. The procedure protocols for 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...a flow rate, a reaction temperature in the reaction chambers, mixing or dilution processes, and the concentrations of the detection reagents. This also applies to a method in which the analytes to be analyzed belong to the same analyte type. In this way, an increase in the sensitivity and specificity of the reactions taking place within a microfluidic device, in particular a cartridge, is achieved.

[0066] A key advantage of determining the concentrations of these hormones is that they are relevant to the female cycle. Measuring their concentration allows for inferences about the cycle, such as the day of the cycle or the fertile days. Furthermore, natural or unhealthy hormone concentrations and fluctuations can be identified, which can provide insights into potential illnesses or phases, such as menopause or favorable training phases for athletes.

[0067] In a further advantageous embodiment, in step b) the sample is allowed to rest and / or gently agitated in the respective reaction chamber for a predetermined time, in particular for 1–5 minutes. An incubation period during which the sample remains stationary or gently agitated in the reaction chamber promotes the binding of the analytes to the respective specific immobilized capture antibodies, thus enabling a very accurate determination of the analyte concentration in the sample.

[0068] Furthermore, in another embodiment, it is advantageous to temperature-control the first and / or the second reaction chamber, in particular to 21–37 °C. The advantage here is that this provides optimal local binding conditions for the binding of the analytes to the respective specific immobilized capture antibodies.

[0069] In a further advantageous embodiment, the process of introducing the sample into a first reaction chamber with a first filter and into at least a second reaction chamber with a second filter in step b) is repeated by introducing the sample in a microfluidic circuit. In this way, the analytes are sequentially bound to the immobilized capture antibodies on the filter membranes iteratively by multiple passes.

[0070] This advantageously increases the binding chances of the analytes to the respective specific immobilized capture antibodies and obtains a geometric array based on filters.

[0071] In an advantageous embodiment, following step c), a recommendation for action and / or information, in particular a phase and / or day in the female cycle and / or a fertile window of a test subject, is calculated and displayed in step d) based on the respective analyte concentrations. The output of the recommendation for action and / or the information is displayed, in particular, on a display unit of the microfluidic device or the analyzer. Alternatively or additionally, the determined concentration of the at least two analytes can also be displayed. Additionally or alternatively, the recommendation for 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.The advantage here is that, instead of a stripe in the reading window of existing lateral flow tests that the test subject has to interpret themselves, they receive the concentration of the analytes and / or information and / or a recommendation for action. This avoids uncertainty for the test subjects and, for example, unintended pregnancies or the unnecessary intake of medication.

[0072] Brief description of the drawing

[0073] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows:

[0074] Fig. 1: schematic representation of a microfluidic device according to the invention in a first embodiment with three filters,

[0075] Fig. 2: a schematic representation of an embodiment of the method according to the invention,

[0076] Fig. 3: the schematic representation of the microfluidic device according to the invention as shown in Figure 1 in various process steps, Fig. 4: the schematic representation of the microfluidic device according to the invention as shown in Fig. 3, which is designed in the form of a cartridge.

[0077] Embodiments of the invention

[0078] Figure 1 shows a first embodiment of a microfluidic device 10 for point-of-care or home diagnostics, for the parallel and multiplex determination of the concentration of three analytes in a sample. The microfluidic device 10 comprises an input chamber 1 and reaction chambers (not shown in Figure 1), each containing a filter 14a, 14b, 14c. A first filter 14a is located in the first reaction chamber, a second filter 14b in the second reaction chamber, and a third filter 14c in the third reaction chamber. The input chamber 1 is located upstream of the reaction chambers. A sample to be analyzed is introduced into the input chamber 1 via a microfluidic inlet 2, shown as an arrow. Specific detection antibodies 3a, 3b, 3c, each with a label 33a, 33b, 33c for readout, are non-immobilized and located upstream of the input chamber 1 in either dry or liquid form.The first analyte-specific detection antibody 3a with the first label 33a binds specifically to a first analyte, the second detection antibody 3b with the second label 33b binds specifically to a second analyte, and the third detection antibody 3c with the third label 33c binds specifically to a third analyte.

[0079] For example, input chamber 1 has a volume ranging from 500 microliters to a few milliliters.

[0080] Furthermore, the input chamber 1 optionally includes a heating device 7, so that the input chamber 1 can be temperature-controlled. In an alternative embodiment, not shown, the detection antibodies 3a, 3b, 3c are not located upstream of the input chamber 1, but are instead, for example, in a reservoir. Alternatively, and also not shown, the microfluidic device 10 does not include an input chamber 1.

[0081] The reaction chambers have dimensions of, for example, 0.5 x 0.5 cm. The material of the reaction chamber side walls is, for example, polycarbonate and / or polypropylene. A first specific capture antibody 5a is immobilized on the first filter 14a of the first reaction chamber, which binds specifically to the first analyte. A second specific capture antibody 5b is immobilized on the second filter 14b of the second reaction chamber, which binds specifically to the second analyte, and a third specific capture antibody 5c is immobilized on the third filter 14c of the third reaction chamber, which binds specifically to the third analyte. Furthermore, the microfluidic device 10 includes, for example, a pre-treatment reagent (not shown) comprising competitive analytes for binding to specific immobilized capture antibodies 5a, 5ab, 5c in the respective reaction chamber to which no analyte has bound.

[0082] Filters 14a, 14b, 14c, for example, are electrically conductive and the reaction chambers comprising them each include two electrodes 8, so that an enzymatic reaction in the respective reaction chamber can be electrically detected.

[0083] Alternatively or additionally, filters 14a, 14b, 14c include an integrated, in particular pH-sensitive, dye, so that an enzymatic reaction in the respective reaction chamber can be detected colorimetrically.

[0084] For pre-storage, the microfluidic device 10 includes, for example, any number of reservoirs, which are not shown in the figures. These reservoirs can contain, for example, buffers or reagents, such as for dilution, staining, or washing of the sample.

[0085] The input chamber 1 and the first, second, and third reaction chambers, each with its respective filter 14a, 14b, 14c, are interconnected via a fluidic network of microfluidic channels 19, in particular in a closed loop. The microfluidic channels 19 are opened and closed via valves 11 (not shown in the figures). The microfluidic device 10 also includes microfluidically connected reservoirs and / or waste containers.

[0086] Furthermore, the microfluidic device 10 includes, for example, a readout unit (not shown in the figures), such as a current / voltage source and / or a camera / LED, for detecting the analytes subsequently bound to the immobilized capture antibodies 5a, 5b, 5c. The microfluidic device 10 also includes, for example, an evaluation unit configured to determine the concentration of the analytes. Finally, the microfluidic device 10 includes, for example, a display unit (not shown in Figure 1).

[0087] The readout unit, the evaluation unit, and the display unit are, for example, 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 and / or evaluation unit and / or display unit located in an analyzer.

[0088] Furthermore, the microfluidic device 10, in particular designed as a cartridge 100, and / or the analyzer comprises, for example, at least one pump, in particular a diaphragm pump.

[0089] Figure 2 shows a flowchart of an embodiment of the inventive method 50 for the parallel, in particular multiplex, determination of the concentration of at least two analytes in a sample, and Figure 3 shows the microfluidic device 10 according to Figure 1 in various process steps. Figure 3 illustrates the detection of three analytes using a sandwich ELI SA.

[0090] As an example for other analytes detectable by sandwich ELISA, in particular peptide hormones, the method according to the invention is described below using LH as the first analyte 4a, FSH as the second analyte 4b and hCG as the third analyte 4c in an exemplary embodiment.

[0091] In step a), for example at home, a defined volume of a biological sample, in particular a urine sample, is introduced, for example by means of a pipette, into the microfluidic device 10, in particular the cartridge 100, via the fluidic inlet 2 into the input chamber 1. This is illustrated in Figure 3 by the analytes 4a, 4b, 4c shown next to the inlet 2.

[0092] In the embodiment shown in Fig. 3, the input chamber 1 contains first detection antibodies 3a with label 33a, which bind specifically to LH as the first analyte 4a in the sample, as well as second detection antibodies 3b with label 33b, which bind specifically to FSH as the second analyte 4b in the sample, and third detection antibodies 3c with label 33c, which bind specifically to hCG as the third analyte 4c in the sample. After binding, LH 4a, FSH 4b, and hCG 4c are bound to the corresponding specific detection antibodies 3a, 3b, and 3c, respectively, and form analyte-detection antibody complexes. This is shown in Figure 3 in the input chamber 1. To accelerate and support this binding, the input chamber 1 is, for example, temperature-controlled.

[0093] In step b), the sample is passed through the first 14a, the second 14b, and the third filter 14c into the first, second, and third reaction chambers. During this process, the LH detection antibody complex binds to the first analyte-specific capture antibody 5a, which is immobilized on the first filter 14a; the FSH detection antibody complex binds to the second specific capture antibody 5b, which is immobilized on the second filter 14b; and the hCG detection antibody complex binds to the third specific capture antibody 5c, which is immobilized on the third filter 14c. LH 4a, FSH 4b, and hCG 4c are then located in a sandwich position between the respective immobilized capture antibody 5a, 5b, 5c and the analyte detection antibody 3a, 3b, 3c with labels 33a, 33b, 33c in a so-called sandwich structure. These are shown in Figure 3 mounted on the respective filter 14a, 14b, 14c.

[0094] To accelerate and support binding, the sample is allowed to stand and / or gently agitated in the reaction chambers for a predetermined time, particularly 1–5 minutes, for example at 21–37°C. This is then advantageously followed by a washing step involving the addition of a washing reagent stored in a reservoir into the reaction chambers.

[0095] Alternatively, and not shown in the figures, the microfluidic device 10 may, for example, not include an input chamber. The sample containing the analytes LH 4a, FSH 4b, and hCG 4c is introduced into the microfluidic device 10 and passed through the reaction chambers with the filters 14a, 14b, and 14c. The analytes LH 4a, FSH 4b, and hCG 4c bind to the specific capture antibodies 5a, 5b, and 5c immobilized on the filters 14a, 14b, and 14c. Subsequently, detection antibodies 3a, 3b, and 3c, labeled 33a, 33b, and 33c, which may be stored upstream in a reservoir, are introduced into the reaction chambers, where they then bind to the filter-bound analytes LH 4a, FSH 4b, and hCG 4c.

[0096] The step of binding the analytes or the detection antibody-bound analytes LH 4a, FSH 4b and hCG 4c Oe (according to the embodiment) to the capture antibodies 5a, 5b, 5c can be repeated as often as desired by passing the sample in a fluidic circuit.

[0097] Advantageously, a washing step is then carried out by adding a washing reagent, which is stored in a reservoir, into the reaction chambers.

[0098] Alternatively or additionally, and not shown in Figures 1, 3 and 4, analytes can be detected using a competitive ELISA. Steroid hormones, in particular estrogen, progesterone and testosterone, can be detected using a competitive ELISA.

[0099] Before the sample is introduced in step a), a competitive analyte to the analyte to be detected is first added to the microfluidic device 10. This analyte binds to the respective analyte-specific capture antibody immobilized on the respective filter. The competitive analyte already carries a label, in particular an enzyme, for subsequent selection.

[0100] In step a), the sample is introduced into the microfluidic device 10, and in step b), the sample is directed into the reaction chambers containing filters 14a, 14b, and 14c with specific immobilized capture antibodies. The analyte to be detected displaces the competing analyte from the analyte-specific immobilized capture antibody and binds to it.

[0101] To accelerate and support binding or displacement, the sample is allowed to stand and / or gently agitated in the reaction chambers for a predetermined time, particularly 1–5 minutes, for example at 21–37°C. This is then advantageously followed by a washing step involving the addition of a washing reagent stored in a reservoir into the reaction chambers, for example, to remove displaced competitive analytes.

[0102] The further procedure is the same for the detection of the analytes in the sandwich ELISA and for the detection of the competitive analytes in the competitive ELISA and is described below for the sandwich ELISA for the example analytes LH 4a, FSH 4b and hCG 4c and for the competitive ELISA in general.

[0103] All detection antibodies 3a, 3b, 3c and / or competitive analytes advantageously carry the same label 33a, 33b, 33c for selection. An enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), serves as the selection label.In step c), a selection reagent for the detection of the labeled detection antibodies 3a, 3b, 3c and / or for the detection of the labeled competitive analytes, in particular from a reservoir of the microfluidic device 10, in particular cartridge 100, is introduced into the reaction chambers. The selection reagent 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 3a, 3b, 3c or of the competitive analyte, for example, horseradish peroxidase, to convert the substrate, thereby inducing, for example, an electrochemical reaction in the form of a redox reaction and / or a color reaction.

[0104] For the detection of a redox reaction, the respective filters 14a, 14b, 14c are electrically conductive and each reaction chamber includes two electrodes 8.

[0105] The electrical measurement starts with the addition of the readout reagent. If LH 4a, FSH 4b and hCG 4c are sandwiched to the filter membrane, or if competitive analytes with labels are bound to the filter membrane, the enzymatic redox reaction starts near the membrane, which is detected by the two-electrode system.

[0106] In an alternative or additional embodiment, the concentration of LH 4a, FSH 4b, and hCG 4c, or the competitive analytes with label, is determined colorimetrically in step c) by detecting a color change in a filter 14a, 14b, 14c, particularly a pH-sensitive filter 14a, 14b, 14c. Here, the enzyme reaction leads, for example, directly—or, in the case of a pH-sensitive filter 14a, 14b, 14c indirectly, via a change in pH caused by the enzyme reaction—to a color change in the dye integrated into the filter 14a, 14b, 14c, so that the color change can be read out colorimetrically. During the readout, the concentration of the bound analytes LH 4a, FSH 4b, and hCG 4c, or the competitive analytes with label, is measured based on the absorption of light in a specific wavelength range.

[0107] A readout unit, in particular a current / voltage source, and / or a camera, detects the electrochemical reaction or the intensity of the color reaction. The more analytes are bound to the immobilized specific capture antibodies in the sandwich ELISA, the higher the detected signal.

[0108] For competitive analytes, the higher the analyte concentration in the sample, the fewer competitive analytes are bound, as the binding sites of many specific capture antibodies immobilized in the reaction chamber are occupied by the analyte. Therefore, the more analyte present in the sample, the lower the detected signal during readout. Conversely, if the analyte concentration is low, many competitive analytes can bind to the specific capture antibodies immobilized in the reaction chamber. A correspondingly high signal is then detected during readout.

[0109] Based on the redox reaction and / or color reaction detected by the readout unit, the concentrations of the hormones LH, FSH and hCG or the competitive analytes are determined using software with a comprehensive evaluation algorithm, in particular based on artificial intelligence.

[0110] Furthermore, the evaluation unit advantageously allows for a comparison of the determined hormone concentrations in the sample with known concentrations, and for an evaluation and classification of these.

[0111] In a further step, the determined concentrations of LH 4a, FSH 4b and hCG 4c are then displayed and / or, based on the classification and evaluation of the respective 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 displayed, for example via a display unit and / or an associated app.

[0112] Depending on the analytes to be detected, sandwich ELISA and competitive ELISA can be combined, i.e., on one of the filters 14a, 14b, 14c, an analyte is detected that can be detected by a sandwich ELISA, and on another filter 14a, 14b, 14c, a different analyte is detected that can be detected by a competitive ELISA.

[0113] The readout unit and / or the evaluation unit and / or the at least one heating element 7 are, for example, part of a microfluidic device 10, which is in the form of a single unit. Alternatively, the microfluidic device 10 is designed as a cartridge 100. This is shown in Figure 4 as an example for the device according to Figure 3. Here, the at least one readout unit and / or the evaluation unit and / or the at least one heating element 7 are not part of the cartridge 100 but are located in a separate analysis device, not shown. The cartridge 100 then has interfaces to this device, which are not shown in the figures. The cartridge 100 may also have other components, not shown.

Claims

Claims 1. Microfluidic device (10), in particular cartridge (100) for point-of-care or home diagnosis, for the parallel, in particular multiplex, determination of the concentration of at least two analytes (4a, 4b, 4c), in particular of different analyte types, in a sample, in particular a urine sample, comprising a first reaction chamber with a first filter (14a) and at least a second reaction chamber fluidically connected to it with a second filter (14b), wherein at least one first specific capture antibody (5a) of a first analyte (4a) to be detected is immobilized on the first filter (14a), and wherein at least one second specific capture antibody (5b) of a second analyte (4b) to be detected is immobilized on the second filter (14b), and further comprising specific detection antibodies (3a) upstream in the microfluidic device (10)., 3b, 3c) for binding to the respective analytes (4a, 4b, 4c) and / or competitive analytes upstream in the microfluidic device (10) for binding to the at least one specific immobilized capture antibody (5a, 5ab,) on the first (14a) and / or on the at least one second filter (14b).

2. Microfluidic device (10) according to claim 1, wherein at least one of the filters (14a, 14b, 14c) is electrically conductive and the reaction chamber comprising it includes two electrodes (8) so that an enzymatic reaction in the reaction chamber can be electrically detected.

3. Microfluidic device (10) according to one of the preceding claims, wherein at least one of the filters (14a, 14b, 14c) comprises an integrated, in particular pH-sensitive, dye, so that an enzymatic reaction in the reaction chamber can be detected colorimetrically.

4. Microfluidic device (10) according to one of the preceding claims, further comprising an input chamber, in particular a heated one. (1) which is located upstream of at least one of the reaction chambers and in particular wherein the specific detection antibodies (3a, 3b, 3c) are located upstream in the input chamber (1).

5. Microfluidic device (10) according to any of the preceding claims, wherein the specific capture antibodies (5a, 5b, 5c) immobilized on the filters (14a, 14b, 14c) are specific for luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) and / or tyrotropin (TSH) and / or human chorionic gonadotropin (hCG), and / or testosterone and / or estrogen and / or progesterone.

6. Microfluidic device (10) according to one of the preceding claims, further comprising at least one electrical and / or colorimetric and / or optical readout unit for detecting the respective analytes (4a, 4b, 4c) bound to the immobilized capture antibodies (5a, 5b, 5c) and an evaluation unit configured to determine the concentration of the at least two analytes (4a, 4b, 4c) by means of an evaluation algorithm, in particular based on artificial intelligence.

7. Microfluidic system comprising a microfluidic device (10) according to any one of the preceding claims 1-5, wherein the microfluidic device (10) is configured as a cartridge (100), and an analyzer comprising an electrical and / or optical readout unit for detecting the respective analytes (4a, 4b, 4c) bound to the immobilized capture antibodies (5a, 5b, 5c) and an evaluation unit configured to determine the concentration of the at least two analytes (4a, 4b, 4c) by means of an evaluation algorithm, in particular based on artificial intelligence, and wherein the analyzer is configured to process the cartridge (100) and / or read out results from it.

8. Method (50) for the parallel, in particular multiplex, determination of a concentration of at least two analytes (4a, 4b, 4c), in particular of different analyte types, in a sample, in particular a urine sample, by means of a microfluidic device (10) according to one of claims 1-6 or by means of a microfluidic system according to claim 7, comprising the following steps a) Providing the sample and introducing a sample volume into the microfluidic device (10), in particular into the input chamber (1). b) Directing the sample into a first reaction chamber with a first filter (14a) and into at least a second reaction chamber with a second filter (14b) and binding the first analyte (4a) to a first specific capture antibody (5a) immobilized on the first filter (14a) and binding the at least one second analyte (4b) to a second specific capture antibody (5b) immobilized on the second filter (14b). c) Determining the concentration of the first filter-bound analyte (4a) and the at least one second filter-bound analyte (4b) and in particular comparing these with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations is carried out in an evaluation unit using an evaluation algorithm, in particular based on artificial intelligence.

9. Method according to claim 8, wherein the determination of the concentration of the analytes (4a, 4b, 4c) in step c) is carried out electrically on the basis of an enzymatic reaction, in particular a redox reaction, detected by means of a two-electrode system in the respective reaction chamber.

10. Method according to one of the preceding claims 8 or 9, wherein the determination of the concentration of the analytes ((4a, 4b, 4c) in step c) is carried out colorimetrically by means of a color change of a filter (14a, 14b, 14c), in particular a pH-sensitive filter (14a, 14b, 14c).

11. Method according to any one of claims 8-10, wherein at least one of the analytes (4a, 4b, 4c) is a peptide hormone, in particular luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH) and / or tyrotropin (TSH) and / or human chorionic gonadotropin (hCG), and wherein at least one of the analytes (4a, 4b, 4c) is a steroid hormone, in particular testosterone and / or estrogen and / or progesterone.

12. Method according to one of claims 8-11, wherein in step b) the sample is allowed to rest for a predetermined time, in particular for 1-5 min. and / or gentle movement of the sample in the respective reaction chamber takes place.

13. Method according to one of claims 8-12, wherein the first and / or the at least one second reaction chamber is at least partially temperature-controlled, in particular to 21-37°C.

14. Method according to any one of claims 8-13, wherein step b) is repeated by guiding the sample in a circuit.

15. Method according to one of claims 8-14, wherein, after step c), in step d), the respective concentration of the analytes (4a, 4b, 4c) 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.

Images