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 multiplex determination of a concentration of n analytes, where n is a natural number >1, in a sample, in particular a urine sample, comprising a reaction chamber (3), in particular a temperature-controlled one, which has a number n of different, in particular magnetic, beads (2a, 2b, 2c) with a successively decreasing diameter, and wherein each bead (2a, 2b, 2c) is coupled to a specific capture antibody (5a, 5b, 5c), wherein at least one first bead (2a) coupled to a first specific capture antibody (5a) has a first diameter, and and wherein at least one further n-th bead (2b, 2c) coupled to an n-th specific capture antibody (5b, 5c) has a smaller diameter than the (n - 1)th bead, wherein the first specific capture antibody (5a) specifically binds a first analyte, and wherein the at least one n-th specific capture antibody (5b, 5c)) specifically binds an n-th analyte, and comprising a number n of microfluidically interconnected, in particular temperature-controlled, retention chambers, each with a retention element (14a, 14b, 14c), wherein the pores of the first retention element (14a) have a first diameter and the pores of the at least one nth retention element (14b, 14c) have a smaller diameter than the (n - 1)th retention element, and wherein the diameter of the at least one first bead (2a) is larger than the diameter of the pores of the first retention element (14a), so that the at least one first bead (2a) can be retained on the first retention element (14a), and wherein the diameter of the at least one n-th bead (2b, 2c) is smaller than the diameter of the pores of the first retaining element (14a), but larger than the diameter of the pores of the n-th retaining element (14b, 14c), such that the at least one n-th bead (2b, 2c) can be retained on the n-th retaining element (14b, 14c).

Description

The present invention relates to a microfluidic device, in particular to a cartridge, for a 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.Prior ArtThe female cycle is structured according to theory in menstrual phase, follicle phase, egg jump and luteal phase. The relative changes of the manipulated variables involved, namely the hormones, include the different phases. The concentration ranges of hormones in blood and urine are known and serve gynecologists as a basis for diagnosis in the case of dysbalances.Depending on the life phase or orientation of life, knowing which cycle phase the female is located is fundamental to the action accordingly, such as for children's wish to women as well as for prevention and cycle awareness.Basal thermometers and cycle apps, partially combined, provide indirect parameters to the status of the female's cycle. The basal temperature only rises shortly before the egg jump, so that the so-called sterile window (~4 days before) cannot be determined. A consequence may be unwanted pregnancies or less chance of becoming pregnant.So-called ovulation tests make possible a hormone measurement, in particular of luteinizing hormone (LH), which represents a rough evidence for the time of the ovulation. However, as in the case of indirect methods, the file window cannot be completely determined, which may result in the sequences already described. Such ovulation tests are present, for example, as test strips in lateral flow format, the sensitivity of which is often low. In addition, as a rule only one analyte is detectable in the sample.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 comprises structures and mechanisms for the manipulation of a sample taken up, in particular passive components such as channels or reaction chambers or also active components such as valves, pumps or mixers. The second main component is a processing unit for controlling the microfluidic sequences in the cartridge, such as, for example, an actuation of the valves or the pumps, and also for detecting constituents of the sample before, during and / or after processing.WO 2014 / 151456 A2 discloses a multi-chamber measurement system for antibody-based determination of various hormones.EP 0751782 A1 discloses an assay for determining hormones of the female cycle in the blood.Disclosure of the InventionAccording to the invention, a microfluidic device, in particular a cartridge, for a 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, having the features of the independent patent claims are provided.This is based in particular on the fact that the microfluidic device, in particular cartridge, is configured for the multiplexed determination of a concentration of n analytes, wherein n is a natural number greater than 1, thus thus at least two analytes in a sample, in particular a biological sample such as, for example, a urine sample or a blood sample.In this case, the microfluidic device, in particular the cartridge, comprises a reaction chamber which has a number n of different beads with a successively decreasing diameter, and wherein each bead is coupled to a specific capture antibody.The first bead coupled to the first specific capture antibody has a first diameter and the at least one further nth bead coupled to an nth specific capture antibody (5a) has a smaller diameter than the (n-1)th bead. The sign "-" represents a minus one. If, for example, two analytes are determined in the sample, then n=2, and the specific second beads coupled to capture antibodies have a smaller diameter than the (2-1)th bead, i.e. the first bead coupled to the first specific capture antibody. If, for example, n=3, the specific third beads coupled to capture antibodies have a smaller diameter than the (3-1)th bead, i.e. the second bead, and so on.A bead can be understood here to mean a microparticle which can be functionalized or coupled, for example with an antibody.The first specific capture antibody specifically binds a first analyte and the at least one nth specific capture antibody specifically binds an nth analyte.The reaction chamber has, for example, a length and a width of 0.5 cm up to 5 cm each and a height of 200 μm to 1 cm and is manufactured, for example, from a polycarbonate (PC), a polypropylene (PP), polystyrene and / or a cycloolefin copolymer (COP) in order to minimize reactions of the analytes on uncoated surfaces.Furthermore, the microfluidic device, in particular the cartridge, comprises a number n of retaining chambers each with a retaining element. In this case, the first retaining chamber is microfluidically connected to the at least one nth retaining chamber. The retaining element is, for example, a microfilter or a microscreen. The diameter of the pores of the first and the at least one n-th retaining element is, for example, in a range from 25 nm to several hundred nm. The retaining element comprises, for example, silica or a polymer- or synthetic resin-based material or a metal, in particular stainless steel, silicon, silicon oxide and / or other nonmagnetic metals.The retaining chambers have, for example, dimensions of 0.5 to 5 cm in diameter and heights of 200 μm to 0.5 cm and are manufactured, for example, from a PC, COP or a coated polymer such as bovine serum albumin (BSA) in order to avoid reactions of the analytes on uncoated surfaces.The retaining chambers have, for example, an inlet above the retaining element laterally in the flow direction and not directly above the surface of the retaining element fixed therein and, for example, an outlet below the filter in the flow direction, in particular on the opposite side of the inlet. This can be realized, for example, via two planes.At least on an upper side, which is located above the retaining element in the flow direction, the retaining chamber advantageously has a transparent, light-transmissive material, for example a transparent PC, COP and / or polystyrene (PS), for reading out.The pores of the first retaining element have a first diameter and the pores of the at least one nth retaining element have a smaller diameter than the (n-1)th retaining element. If n is two, for example, the pores of the second retaining element have a smaller diameter than the (2-1)th retaining element, that is to say the first retaining element. If n is three, for example, the pores of the third retaining element have a smaller diameter than the (3-1)th retaining element, that is to say the second retaining element.Furthermore, the diameter of the at least one first bead is greater than the diameter of the pores of the first retaining element, such that the at least one first bead is retained on the first retaining element. Furthermore, the diameter of the at least one nth bead is smaller than the diameter of the pores of the (n-1)th retaining element, but larger than the diameter of the pores of the nth retaining element, so that the at least one nth bead is retained on the nth retaining element.It is advantageous here that a very accurate measurement of the concentration of the analytes, in particular of different types of analytes, such as, for example, different types of hormones, in particular peptide hormones and steroid hormones, in a sample is possible at the point of care or conveniently at home for an on-site diagnosis.The respective analytes are detectable specifically and sensitively, so that their concentrations can be determined with precision, as in laboratory analysis at the physician or in the central laboratory. The measurement is carried out in multiplex, since the n analytes can be captured simultaneously, in particular read out simultaneously, and the concentrations of the analytes can be determined in parallel and in the same way. In this way, both time until the result is obtained and practical working time, working steps, containers, in particular reservoirs, and the reagents themselves are saved.It is also particularly advantageous that the concentration of the analytes can be determined at the point of care or at home, i.e. very easily, conveniently and user-oriented. For this purpose, for example, a urine sample or a saliva sample is used. It is advantageous here that this can be obtained without pain and anxiety, in a simple, non-invasive way. Alternatively, however, a blood sample can also be used, for example.Compared to a lateral flow format, the microfluidic environment increases both the sensitivity, the specificity and the degree of multiplexing and also saves reagents compared to individual tests. In addition, this allows tempering and dilution and washing steps.It has also proven advantageous that the sample can be prepared for the analysis, for example, by automated dilution on the microfluidic device, in particular cartridge.The reaction chamber and the retaining chambers are connected to one another via a fluidic network, wherein a sample is first introduced into the reaction chamber and then into the respective retaining chambers with the retaining elements starting with the first retaining chamber with the first retaining element with the largest pore diameter and finally into the next retaining chamber with the retaining element with the next smaller pore diameter. In a preferred embodiment, the retaining chamber with the retaining element with the smallest pore diameter is in turn fluidically connected to the reaction chamber, so that the sample can be guided in a circuit. In an alternative embodiment, after passing the retaining chambers, the sample enters a waste container. Fluids, in particular reagents, samples and buffers, are pressure-driven, for example by means of membrane pumps and / or by means of gravity and / or by means of charge differences transported through the microfluidic network of the microfluidic device or of the cartridge.The reaction chamber and / or the retaining chambers can be temperature-controlled, in particular heated, for example, at least in regions, so that optimum binding conditions for binding the analytes to the respective capture antibodies are present in the reaction chamber and / or so that optimum binding conditions for binding detection antibodies to a further binding site of the analyte and / or for binding competitive analytes to the capture antibodies are present in the retaining chambers.The microfluidic device according to the invention is configured, for example, as a lab-on-a-chip cartridge, referred to below as a cartridge. The cartridge comprises interfaces to a further unit, for example an analysis device, and can comprise further components.Further advantageous embodiments of the microfluidic device are evident from the dependent claims.In a further advantageous embodiment, the antibody-coupled beads comprise a magnetic material, in particular gold, iron and / or platinum, and the retaining chambers are at least partially designed magnetically in a region which is located below the retaining element in the direction of flow. Alternatively, a magnet is disposed downstream of the retention chamber.It is advantageous here that in this way the magnetic antibody-coupled beads are pulled in the direction of the magnetically embodied region of the retaining chamber or toward the magnet under the retaining chamber and collect at a location on the retaining element if the diameter of the bead is greater than the pore diameter of the retaining element. This results in a concentration of the signal intensity in a smaller space, so that during the readout, instead of a signal intensity distributed over the entire surface of the retaining element, a large luminous or color point can be detected in the form of individual small luminous points. This increases the sensitivity. In addition, the readout unit, in particular the camera, can be of simpler construction. If the diameter of the bead is smaller than the pore diameter of the retaining element, the antibody-coupled bead is pulled through it. In an embodiment described below, in which the sample or the analyte is conveyed in a circuit several times via the retaining chambers, the magnet also allows better binding of the analyte to the capture antibodies and the detection antibody to the analyte and / or the competitive analyte to still free specific capture antibody concentrations, because a spatial concentration takes place.Furthermore, in a particularly advantageous embodiment, the specific capture antibodies coupled to the beads are specific for the luteinizing hormone (LH) and / or the follicle stimulating hormone (FSH) and / or tyreotropin (TSH) and / or the human chorionic gonadotropin (hCG) and / or the anti-Muller hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.In an advantageous embodiment, the capture antibodies coupled to the beads are at least specific for LH, FSH, hCG, estrogen and progesterone.These hormones particularly well reflect the cycle status / fertility status of a female, so that by means of these, for example, the entire fertility window of a probandin can be determined very precisely. Further advantages of the analysis of these hormones are listed in the further course of the application.Furthermore, in an advantageous embodiment, it is provided that the microfluidic device or the cartridge comprises at least one reservoir, in which at least one detection reagent comprising specific detection antibodies, which bind to the respective analytes and / or competitive analytes for binding to optionally still free specific capture antibodies, are arranged upstream. The detection antibodies and / or the competitive analytes bear a label for readout. Additionally or alternatively, at least one washing solution and / or at least one readout reagent and / or at least one buffer is placed in front of the at least one reservoir for dilution. By means of a microfluidic connection from the at least one reservoir to a first retaining chamber and / or to the reaction chamber, the reagents stored can be conducted thereto.In one embodiment, the microfluidic device comprises at least one electrochemical and / or optical readout unit for detecting the respective n analytes bound to the bead-coupled specific capture antibodies.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.For example, when using a readout unit in comparison with lateral flow tests without a readout unit, it is advantageous that the sensitivity is increased.The read-out unit is mounted movably, for example, so that it is movable towards the retaining chambers during the read-out. Alternatively, the read-out unit is configured to read out all retaining chambers simultaneously. Further alternatively, a readout unit is provided for each retaining chamber. At least the upper side, i.e. the side of the retaining chamber which is situated above the retaining element as seen in the direction of flow, is made of a transparent material, for example of a transparent PC, COP, glass and / or polystyrene, so that the coupled analytes retained on the retaining element can be read out.Furthermore, the microfluidic device comprises an evaluation unit configured to determine the concentration of the n analytes by means of an evaluation algorithm, in particular based on artificial intelligence. In particular, the evaluation unit is further configured to compare the determined concentrations of the n analytes with known natural concentrations of the respective analytes in order to use these as the basis for the evaluation. Thus, for example, deviations from natural concentrations of the respective analytes can also be detected and / or natural or non-natural fluctuations of the analytes can be detected, for example natural or non-natural fluctuations of hormones. It is advantageous here that the software, given a sufficient data position of a user, determines, for example, deviations from the individual hormone level over the cycle. Furthermore, it is advantageous that an overall image of all specific concentrations of the analytes is thus determined in comparison to the prior art, where this is often only possible at certain points for individual analytes. Advantageously, based on this, a recommendation for action and / or information is output to the user / the user.It is advantageous that the use of artificial intelligence allows quantification of the analytes and also allows evaluation with customer-oriented recommendation for action and / or the output of information, instead of a test strip result to be interpreted by itself in a readout window.In particular, the microfluidic device further comprises a display unit for this purpose, for example a display on which the output recommendation for action and / or information and / or the determined analyte concentrations are 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 output to the user / the user via the app.In an advantageous embodiment, the microfluidic device comprises at least one temperature control device for temperature control, in particular heating, of the reaction chamber and / or of the retaining chambers. The temperature control device is, for example, a heater and / or a Peltier element.The microfluidic device is, for example, a one-piece measuring device in the size of a smartphone, which can be transported conveniently, for example in a handbag.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 analytical device. The analytical device comprises an optical and / or electrochemical readout unit, in particular a camera and / or a luminometer, for detecting the respective n analytes bound to the specific bead-coupled capture antibodies, and an evaluation unit configured to determine the concentration of the n analytes by means of software comprising an evaluation algorithm, wherein the analytical device is configured to process the cartridge and / or to read out results.The readout unit is mounted movably, for example, so that it can be moved towards each retaining chamber during the readout. Alternatively, the reading unit is designed to read all retaining chambers simultaneously.Further explanations already mentioned above with respect to the readout unit apply here accordingly.The evaluation unit is advantageously programmed accordingly together with a processor in the analysis device. The further explanations already mentioned above with respect to the evaluation unit apply here accordingly.Furthermore, the analysis device comprises in particular a display unit to which the above-mentioned explanations also apply and also with respect to the app mentioned there. In addition, the analysis device comprises an optional temperature control device, to which the already mentioned explanations apply as well. Furthermore, the analytical device comprises, for example, a magnet.The cartridge is designed, for example, as a disposable part, while the analytical device is, for example, a multiple use unit.The analytical device has, for example, the size of a smartphone, which can be transported conveniently, for example in a hand bag. The cartridge is smaller compared to the analysis device and has, for example, the size of a softwood box.The analytical device (processing unit) and the cartridge can be designed, for example, as described in DE102016222072A1 or DE102016222075A1 and the cartridge can be processed accordingly.In a further embodiment of the system, both the microfluidic device, in particular cartridge, and the analysis device comprise an optical and / or electrochemical readout unit and / or an evaluation unit and / or a temperature control device and / or a magnet. In this case, the read-out and / or evaluation units can complement one another, for example.The invention further relates to a method for the multiplex determination of a concentration of n analytes, wherein n is a natural number >1, in a sample, in particular in a biological sample such as, for example, a urine sample by means of the microfluidic device or by means of the microfluidic system, with the following steps:a) providing the sample and introducing a particularly defined sample volume into the microfluidic device.The sample, for example a urine sample, is collected, for example, in a vessel and a particularly defined sample volume is introduced, for example, by a user or a user, into the microfluidic device, in particular cartridge, for example via a microfluidic inlet, for example, by means of a pipette or another aid. In the case that the concentration of the sample is too high, it is diluted, in particular automatically, for example, so that the later readout can take place reliably and undisturbed.It is particularly advantageous that the sample is taken at home or at the point of care and analyzed, which saves a lot of time.It is also advantageous here that, if the sample is a urine or saliva sample, it can be obtained without pain and anxiety, in a simple, non-invasive way. Alternatively, a blood sample may be used, for example.The input of a defined sample volume is particularly advantageous since standardization is possible in this way.b) Guiding the sample into a reaction chamber (3), in particular a temperature-controlled reaction chamber, and binding the first analyte to the first specific capture antibody (5a) coupled to the first bead to form a first bead antibody analyte complex, and binding the at least one n-th analyte to the n-th specific capture antibody coupled to the n-th bead to form an n-th bead antibody analyte complex. It is advantageous here that only the appropriate analyte can bind to the specific capture antibody and thus a very specific and accurate result can be obtained. In addition, the analytes can be determined in this way in multiplex.Advantageously, in step b) the sample is allowed to stand for a predetermined time, in particular for 1-15 min. Alternatively, the sample is gently moved in the reaction chamber. An incubation period in which the sample remains resting or gently moved in the reaction chamber promotes the binding of the analytes to the respective specific bead-coupled capture antibodies, so that a very accurate determination of the concentration of the analytes in the sample can be made.c) Directing the sample with the first and the at least one nth bead antibody analyte complex into the first retention chamber and size selective retention of the first bead antibody analyte complex on the first retention element, and passing the first retention element through the at least one nth bead antibody analyte complex.d) Directing the sample with the at least one nth bead antibody analyte complex into the nth retention chamber and size selective retention of the at least one nth bead antibody analyte complex on the nth retention memberIt is advantageous in steps c) and d) that in this way a very good separation of the various analytes in the sample is achieved, as a result of which a very accurate conclusion can finally be drawn about the concentration of the analytes. It is also particularly advantageous that, by such a separation or separation of the analytes in multiplex, the same readout unit can be used for all n analytes for the later readout.Advantageously, steps c) and d) are repeated by pumping the sample in a circuit, in particular with a defined service life, in particular for 0.1-1 min, after each pump cycle.Advantageously, the binding chances of still unbound analytes to the respective specific bead-coupled capture antibodies are thus increased.Advantageously, after step d), a washing step is carried out by adding a washing reagent and flushing it circularly through the reaction chamber and the retaining chambers. The washing reagent is advantageously placed upstream, in particular in a reservoir of the microfluidic device, in particular of the cartridge.e) supplying a specific detection reagent, in particular from a reservoir, and circularly guiding it through the retaining chambers, wherein the specific detection reagent comprises specific detection antibodies with a label, in particular an enzyme, and binding these to the respective analytes of the bead antibody analyte complexes and / or wherein the specific detection reagent comprises competitive analytes with a label, in particular an enzyme, and binding these to optionally still free specific capture antibodies. Competitive analytes are artificial analytes to the analytes to be detected.If the specific bead-coupled capture antibodies already bear a label for readout, step e) is not required.All detection antibodies and / or competitive analytes advantageously bear the same label for the readout. Optionally, one or more detection antibodies can also be present without labels or, for example, with a biotin label. Then, for example, a species-specific secondary antibody is necessary which binds the detection antibody and carries the label for readout, or, in the case of the biotin label, a streptavidin-enzyme complex which binds to the biotin and carries the label for readout. The label used for the readout is, for example, an enzyme, for example horseradish peroxidase (HRP) or alkaline phosphatase (AP).Advantageously, after the addition of the detection reagent, at least one further washing step takes place analogously to the already described washing step in order to wash out unbound detection antibodies and / or unbound competitive analytes. The washing reagent is advantageously placed upstream, in particular in a reservoir of the microfluidic device and in particular of the cartridge.f) adding a readout reagent, in particular from a reservoir of the microfluidic device or of the cartridge. The readout reagent for a detection of the labeled detection antibodies and / or the competitive analyte is, for example, a substrate such as TMB (3,3',5,5'-tetramethyl benzidine) and causes the label, for example the enzyme of the detection antibody or the competitive analyte, to react the substrate, causing a color reaction and / or a chemiluminescent reaction in the form of an emission of electromagnetic radiation in the range of ultraviolet and visible light, and / or a fluorescent reaction in the form of an emission of electromagnetic radiation.A reaction catalyzed by horseradish peroxidase can be read out, for example, colorimetrically, chemifluorescently or chemiluminescently. A reaction caused by the alkaline phosphatase can be read out colorimetrically or chemiluminescenceally, for example. A readout unit, in particular a camera, records the intensity of the color reaction or the density of the generated dye and / or of the emitted electromagnetic radiation.In the case of the competitive analytes, it is the case that if there is a large amount of analyte in the sample, few competitive analytes are bound, since the binding sites of many specific capture antibodies, to which the competitive analytes can then bind, are already occupied by the analyte. During the readout, a low signal is thus detected. If there is little analyte in the sample, the binding sites of many specific capture antibodies remain free, to which the competitive analytes can then bind. During the readout, a high signal is then detected.It is advantageous that for the detection of different types of analytes, wherein in one embodiment at least one type of analyte is detectable by means of labeled detection antibodies and at least one type of analyte is detectable by means of competitive analytes, and thus the intermediate steps are different, which the readout step can nevertheless be carried out by the same readout system with addition of the same readout reagent and detectable by means of the same readout unit. In this way, a high degree of multiplex of analytes is possible. In addition, costs are saved since only one readout system, for example a camera with a filter, is required.Following step f), at least one washing step analogous to the washing steps described above is advantageous.g) determining the concentration of the n analytes on the basis of the detected reaction, in particular the color and / or radiation intensity, and in particular comparing these with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations takes place in the evaluation unit with the aid of an evaluation algorithm, in particular based on artificial intelligence.It is optionally advantageous to prepare the sample for the method before step a) by, depending on the sample, for example separating interfering substances and particles, for example separating particulate matter by means of a filter or, for example, in the case of urine samples, for example performing a plasma separation by means of a centrifuge or, in the case of blood samples, for example.Furthermore, it is advantageous in one embodiment if in steps b) and / or c) and / or d) at least one temperature control of the reaction chamber and / or of the retaining chambers takes place in regions, in particular at 21-40° C. The temperature control or heating takes place over the entire chamber or in individual regions thereof.It is advantageous here that optimal local binding conditions for binding the analytes to the respective specific bead-coupled capture antibodies are present on the temperature side in this way and, when the sample is circulated, the detection antibodies also bind to the analytes and / or the competitive analytes bind to the specific capture antibodies.In a particularly advantageous embodiment of the method, the n analytes are hormones, in particular the luteinizing hormone (LH) and / or the follicle stimulating hormone (FSH) and / or tyreotropin (TSH) and / or the human chorionic gonadotropin (hCG) and / or the anti-Muller hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.It is advantageous in determining the concentrations of these hormones that these are relevant hormones of the female cycle. The measurement of their concentration allows conclusions to be drawn about these, such as the day in the female cycle or the fruitable days. Furthermore, natural or unhealthy hormone concentrations and fluctuations can be determined, which allow conclusions to be drawn about possible diseases or phases, such as, for example, the change years or favorable training phases for sportsists.In an 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.These hormones particularly well reflect the cycle status of a female, so that by means of these, for example, the entire fertility window of a probandin can be determined very precisely.In an advantageous embodiment, after step g), in a step h) the respective concentration of the n analytes is output and / or, on the basis of the concentration of the n analytes, a recommendation for action and / or information, in particular a phase and / or a day in the female cycle and / or a fatiguable window of a probandin is calculated and output. The output of the recommendation for action and / or of the information is displayed in particular on a display unit of the microfluidic device or of the analysis device. Additionally or alternatively, the recommendation for action and / or the information and / or the determined analyte concentrations are transmitted to an associated app and output to the user / the user via the app. It is advantageous here that a probandin, instead of a strip to be interpreted by itself in the readout window of existing lateral flow tests, receives a concentration of the analytes and / or an item of information and / or a recommendation for action on his / her smartphone or the display unit, in particular a display. This prevents uncertainties in the test subjects and, for example, unwanted pregnancy or unnecessary intake of active ingredients and increases the chances for pregnancy in the event of a wish for children.BRIEF DESCRIPTION OF THE DRAWINGEmbodiments 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 : shows the schematic illustration of a microfluidic device according to the invention in a first embodiment before method start, FIG. 2 : shows the schematic illustration of the microfluidic device according to the invention according to FIG. 1 during the method execution, FIG. 3 : shows the schematic illustration of the device according to the invention according to FIG. 2, which is designed in the form of a cartridge, and FIG. 4 : shows the schematic representation of an exemplary embodiment of the method according to the invention.Embodiments of the InventionFIG. 1 shows a first embodiment of the microfluidic device 10 according to the invention. This can be designed as a cartridge 100. By means of the microfluidic device 10 in FIG. 1, the concentrations of three analytes, i.e. n=3, in a sample can be determined in multiplex. in the following this is described by way of example for all analytes on the basis of the peptide hormones LH and FSH and the steroid hormone progesterone. The microfluidic device 10 comprises a reaction chamber 3 in which a first bead 2 acoupled to a first specific capture antibody 5 aand a second bead 2 bcoupled to a second specific capture antibody 5 band a third bead 2 ccoupled to a third specific capture antibody 5 care arranged upstream in a mix.The first bead 2a has a diameter which is larger than the diameter of the second bead 2b. The diameter of the second bead 2b is in turn larger than the diameter of the third bead 2c.The first specific capture antibody 5a specifically binds to LH, the second specific capture antibody 5b specifically binds to FSH, and the third capture antibody 5c specifically binds to progesterone.The reaction chamber has, for example, a length and a width of 0.5 cm up to 5 cm each and a height of 300 μm to 1 cmThe reaction chamber 3 is microfluidically connected to a first retaining chamber with an integrated first retaining element 14 a. The first retaining chamber is microfluidically connected to a second retaining chamber with a second retaining element 14 band the second retaining chamber is microfluidically connected to a third retaining chamber with a third retaining element 14 c. In FIG. 1, only the retaining elements 14 a, 14 b, 14 care illustrated, but not the retaining chambers. The retaining elements 14 a, 14 b, 14 care, for example, microfilters or microscreens made of a silica-based material. The pores of the first retaining element 14a have a larger diameter than the pores of the second retaining element 14b and the pores of the second retaining element 14b have a larger diameter than the pores of the third retaining element 14c.The diameter of the pores of the first, second and third retaining members 14 a, 14 b, 14 cis, for example, in a range of 25 nm to several hundred nm. The retaining chambers have dimensions of, for example, 0.5 cm to 5 cm in diameter and heights of 200 μm to 0.5 cm and are manufactured, for example, from a PC, COP and / or PS.Further, the diameter of the first bead 2a is larger than the diameter of the pores of the first retaining member 14a, so that the first bead 2a is retained on the first retaining member. Furthermore, the diameter of the second bead 2 bis smaller than the diameter of the pores of the first retaining element 14 a, but larger than the diameter of the pores of the second retaining element 14 b, so that the bead 2 bis retained on the second retaining element 14 b. Further, the diameter of the third bead 2c is smaller than the diameter of the pores of the second retaining member 14b but larger than the diameter of the pores of the third retaining member 14c, so that the third bead 14c is retained on the third retaining member.The third retaining chamber is microfluidically connected to the reaction chamber 3 so that a fluid can be circulated through the microfluidic device 10.The sum of all microfluidic connections or channels forms a microfluidic network 19.Furthermore, the microfluidic device 10 or the cartridge comprises at least one reservoir 13, in which at least one detection reagent 55, as shown in FIG. 1, and / or at least one washing solution and / or at least one readout reagent and / or at least one buffer for dilution is arranged upstream. Advantageously, the microfluidic device 10 comprises a plurality of reservoirs 13. Furthermore, the microfluidic device has, for example, a waste container not shown in FIG. 1, for example for storing fluids no longer required. The at least one reservoir 13 and / or the waste container are fluidically connected to the retaining chambers 14 a, 14 b, 14 cand / or the reaction chamber 3 via the fluidic network 19.The microfluidic channels 19 are opened and closed via valves 11. The direction of flow through the microfluidic device is shown in FIG. 1 by means of small arrows on the microfluidic channels.The retaining chambers have, for example, an inlet in the flow direction above the respective retaining element 14 a, 14 b, 14 considence and not directly above the surface of this one inlet and, for example, an outlet in the flow direction below the filter, in particular on the opposite side of the inlet. This can be realized, for example, via two planes. This is not shown in FIG. 1.At least on an upper side, which is located above the retaining element in the flow direction, the retaining chamber has a transparent material, for example a transparent PC, COP, PS and / or glass, for reading out.The microfluidic device 10 comprises a microfluidic inlet 1, illustrated as an arrow in FIG. 1, via which a sample, for example a urine sample, can be introduced into the device 10.Furthermore, at least one region of the reaction chamber 3 and / or of the retaining chambers 14 a, 14 b, 14 cmay be temperature-controllable. For this purpose, the microfluidic device 10 or, if it is configured, for example, as a cartridge 100, comprises an analytical device not shown in the figures, a temperature control device 7 shown only symbolically in FIG. 1, for example a heater or a Peltier element. If the microfluidic device 10 is designed as a cartridge 100, the cartridge 100 comprises interfaces to the temperature control device 7 arranged on the analysis device, for example.Advantageously, the antibody-coupled beads 2 a, 2 b, 2 ccomprise a magnetic material, in particular gold, iron and / or platinum, which can additionally be functionalized, for example, with dextran derivatives such as, for example, carboxymethyldextran (CMD) and / or aminodextran (AMD), for a better biofunctionality. The retaining chambers are, for example, at least partially designed magnetically in a region which is located below the retaining element 14 a, 14 b, 14 cin the flow direction. Alternatively, a magnet 8 is arranged below the corresponding retaining chamber in the direction of flow and / or in a position located below the retaining chamber in an analysis device. This is only shown symbolically in FIG. 1. The direction of flow through the microfluidic device 10 is indicated by means of small arrows on the microfluidic channels of the microfluidic network 19.Furthermore, FIG. 1 likewise only represents symbolically a readout unit 9 in the form of a camera 9, by means of which the hormones bound to the specific capture antibodies 5 a, 5 b, 5 cmay be read out on the retaining elements 14 a, 14 b, 14 cin the further course. The camera 9 is mounted movably, which is to be illustrated by the arrows 9 b. In FIG. 1, the readout range 9 aof the camera 9 is intended to show that all retaining elements 14 a, 14 b, 14 care read out by a camera 9. The readout unit 9 is, for example, a part of the microfluidic device 10 itself or, if the microfluidic device 10 is, for example, embodied as a cartridge 100, the cartridge 100 comprises an interface to a readout unit 9 located in an analytical instrument.FIG. 2 shows the microfluidic device according to FIG. 1 during the execution of the method. The beads 2 a, 2 b, 2 ccoupled to a specific capture antibody 5 a, 5 b, 5 care already brought into contact with the sample, so that bead-antibody-hormone complexes have already formed, which circulate through the microfluidic device 10. First bead-antibody-LH complexes are retained on the first retaining element 14a. Second bead-antibody-FSH complexes are retained on the second retaining element 14 band third bead-antibody-progesterone complexes are retained on the third retaining element 14 c. For the readout by means of the readout unit, which is designed as a camera 9 in FIG. 1, the latter is mounted movably, for example, so that it is movable towards the retaining chambers 14 a, 14 b, 14 cwhen being read out. This is illustrated by the arrows 9 bto the right and left of the camera 9, for example.Alternatively, the reading unit is configured to read all retaining chambers 14 a, 14 b, 14 c simultaneously. Further alternatively, an extra readout unit is provided for each retaining chamber 14a, 14b, 14c. At least the upper side, i.e. the side of the retaining chamber which is situated above the retaining element 14 a, 14 b, 14 c, as seen in the direction of flow, is made of a transparent material, for example of a transparent PC, COP, glass and / or PS, such that the coupled analytes retained on the retaining element can be read out. In FIG. 2, the readout region 9 aof the camera 9 is intended to show that all retaining elements 14 a, 14 b, 14 care read out by a camera 9, but this can be, but need not be, the same camera.Furthermore, the microfluidic device 10 comprises, for example, an evaluation unit not shown in the figures, or, if the microfluidic device 10 is designed as a cartridge 100, the cartridge 100 comprises at least one interface to at least one evaluation unit located in an analytical instrument. The evaluation unit is configured to determine a concentration of n analytes.In addition, the microfluidic device 10 comprises, for example, a display unit not shown in the figures, 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 analytical instrument.In addition, the microfluidic device, in particular configured as a cartridge, or the analysis device comprises, for example, at least one pump, in particular a membrane pump.Thus, the at least one readout unit 9 and / or the evaluation unit and / or the at least one temperature control device 7 are, for example, part of a microfluidic device 10, which is present in the form of a one-piece device or, alternatively, the microfluidic device 10 according to FIG. 2 is designed as a cartridge 100-as shown in FIG. 3-, wherein the at least one readout unit 9 and / or the evaluation unit and / or the at least one temperature control device 7 are not part of the cartridge 100 and are present in a separate, not shown analysis device. The cartridge then has interfaces to it, not shown in the figures. The cartridge 100 can have further components, not shown.FIG. 4 shows a flow diagram of an exemplary embodiment of the method 50 according to the invention for the multiplex determination of a concentration of three analytes present in a sample at the point of care or at home.Representative of other or further analytes, the method according to the invention is described below by way of example for the hormones LH as the first analyte, FSH as the second analyte and progesterone as the third analyte.In a step a), a defined volume of a biological sample, in particular a urine sample, of a, for example, female probandin, is introduced into the microfluidic device 10, or the cartridge, via the fluidic inlet 1, for example by means of a pipette.In a step b), the sample is fed into the reaction chamber 3, in particular temperature-controlled to 21-40° C., in which bead-coupled specific capture antibodies 5 a, 5 b, 5 care arranged upstream.For a predetermined time, in particular for 1-15 min, the sample is allowed to stand or the sample is moved gently in the reaction chamber 3.The first specific capture antibody 5a coupled to the first bead 2a specifically binds to the LH to form a first bead-antibody-LH complex. The second specific capture antibody 5b coupled to the second bead 2b specifically binds to the FSH to form a second bead antibody-FSH complex, and the third specific capture antibody 5c coupled to the third bead 2c specifically binds to progesterone to form a third bead antibody-progesterone complex. For the sake of simplicity, these will be referred to below as the first complex, the second complex and the third complex.In a further step c), the sample with the at least one first, second and third complex is conducted into the first retaining chamber with the first retaining element 14. On the first retaining element 14 a, the first complex is retained due to the size due to the diameter of the first bead 2 a. The second and third complexes can pass through the first retaining element 14 a.In a further step d), the sample with the second and the third complex is conducted into the second retaining chamber with the second retaining element 14 b. On this latter, the second complex is retained due to the size of the diameter of the second bead 2 b. The third complex passes through the second retaining element 14 b.Further, the sample with the third complex is introduced into the third retaining chamber with the third retaining member 14c. On this, the third complex is retained due to the size due to the diameter of the third bead 2c. Thus, the LH from the sample is on the first retention element in the first complex, the FSH on the second retention element in the second complex, and the progesterone on the third retention element in the third complex.Steps c) and d) are repeated by circulating the sample in a circuit, in particular with a defined service life for 0.1-1 min, after each pump cycle.In steps c) and d), the first, second and third retaining chambers are advantageously tempered, in particular to 21-40° C. ° C.This is followed by a washing step by addition of a washing reagent stored in a reservoir 13. This is flushed circularly through the reaction chamber and the retaining chambers.Subsequently, in a step e), a specific detection reagent 55, in particular one placed upstream in a reservoir 13, is added, wherein the specific detection reagent 55 comprises specific detection antibodies 55 awith a label 55 band / or competitive analytes with a label. The specific detection antibodies 55a specifically bind to the peptide hormones LH and FSH in the respective bead antibody-hormone complex. Competitive analytes for progesterone bind to the capture antibodies (5a, 5ab, 5c), which are optionally still free and are specific for progesterone.Advantageously, all detection antibodies 55 aand / or competitive analytes bear the same label 55 bfor later readout. The label used is, for example, an enzyme, for example the HRP.Advantageously, after the addition of the detection reagent 55, at least one further washing step takes place, which takes place analogously to the above-described washing step.Subsequently, in a step f), a readout reagent, in particular from a reservoir 13 of the microfluidic device 10 or the cartridge 100, is added. The readout reagent for the detection of the detection antibodies 55a with the label 55b and / or the competitive analytes with the label is, for example, a substrate such as TMB (3,3',5,5'-tetramethyl benzidine) and causes the label 55b, the detection antibody 55a and / or the competitive analyte to react the substrate, causing a color reaction and / or a chemiluminescent reaction in the form of an emission of electromagnetic radiation in the range of ultraviolet and visible light, and / or a fluorescent reaction in the form of an emission of electromagnetic radiation. The reaction catalyzed by the HRP can be read, for example, colorimetrically, chemifluorescently or chemiluminescently.A readout unit 9, in particular a camera 9, records the intensity of the color reaction or the density of the generated dye and / or of the emitted electromagnetic radiation.Following step g), at least one washing step analogous to the washing steps described above is advantageous.In a subsequent step g), the concentrations of the hormones LH, FSH and progesterone are determined on the basis of the color and / or radiation intensity detected by the readout unit 9 by means of software comprising an evaluation algorithm, in particular based on artificial intelligence.In addition, the evaluation unit advantageously compares the determined hormone concentrations in the sample with known concentrations and evaluates and classifies them.In a step h), for example, the respective concentration of the hormones LH, FSH and progesterone is emitted. On the basis of the classification and evaluation of the respective hormone concentrations, for example, a recommendation for action and / or information, in particular a phase and / or a day in the female cycle and / or a fatigued window of a subject is output, for example, via a display unit and / or an associated app. Furthermore, natural or unhealthy hormone concentrations and fluctuations can be determined, which allow conclusions to be drawn about possible diseases or phases, such as, for example, the change years or favorable training phases for sportsists.References included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedWO 2014 / 151456 A2

[0007] EP 0751782 A1

[0008] DE 102016222072A1

[0053] DE 102016222075A1

[0053]

Claims

Microfluidic device (10), in particular cartridge (100) for a diagnosis at the point of care or at home, for the multiplexed determination of a concentration of n analytes, wherein n is a natural number >1, in a sample, in particular a urine sample, comprising a reaction chamber (3), which can be controlled in particular and has a number n of different beads (2a, 2b, 2c) with a successively decreasing diameter, and wherein each bead (2a, 2b, 2c) is coupled to a specific capture antibody (5a, 5b, 5c), wherein at least one first bead (2a) coupled to a first specific capture antibody (5a) has a first diameter, and wherein at least one further bead (2a) coupled to an nth specific capture antibody (5b, 5c) coupled n-th bead (2b, 2c) has a smaller diameter than the (n-1)th bead, wherein the first specific capture antibody (5a) specifically binds a first analyte, and wherein the at least one n-th specific capture antibody (5b, 5c)) specifically binds an n-th analyte, and comprising a number n of microfluidically connected, in particular temperature-controllable, retention chambers each having a retention element (14a, 14b, 14c), wherein the pores of the first retention element (14a) have a first diameter and the pores of the at least one n-th retention element (14b, 14c) have a smaller diameter than the (n-1)th retention element, and wherein the diameter of the at least one first bead (2a) is larger than the diameter of the pores of the first retaining element (14a), such that the at least one first bead (2a) is retainable on the first retaining element (14a), and wherein the diameter of the at least one nth bead (2b, 2c) is smaller than the diameter of the pores of the (n-1)th retaining element (14a), but larger than the diameter of the pores of the nth retaining element (14b, 14c), such that the at least one nth bead (2b, 2c) is retainable on the nth retaining element (14b, 14c).Microfluidic device (10), in particular cartridge (100), according to claim 1, wherein the specific capture antibodies (5a, 5b, 5c) are furthermore each coupled to a label, in particular an enzyme, for reading out.Microfluidic device (10), in particular cartridge (100), according to one of the preceding claims, wherein the beads (2a, 2b, 2c) comprise a magnetic material, in particular gold, iron, graphite and / or platinum, or are coated with such a material, and wherein the retaining chambers are at least partially designed magnetically in a region which is located below the respective retaining element (14a, 14b, 14c) in the flow direction, or wherein a magnet (8) is arranged below the respective retaining chamber in the flow direction.Microfluidic device (10), in particular cartridge (100), according to one of the preceding claims, wherein the specific capture antibodies (5a, 5b, 5c) coupled to the beads (2a, 2b, 2c) are specific for the luteinizing hormone (LH) and / or the follicle stimulating hormone (FSH) and / or tyreotropin (TSH) and / or the human chorionic gonadotropin (hCG) and / or the anti-Muller hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.Microfluidic device (10), in particular cartridge (100), according to claim 4, wherein the capture antibodies (5a, 5b, 5c) coupled to the beads (2a, 2b, 2c) are at least specific for the luteinizing hormone (LH), the follicle stimulating hormone (FSH), the human chorionic gonadotropin (hCG), estrogen and progesterone.Microfluidic device (10), in particular cartridge (100), according to one of the preceding claims, wherein the microfluidic device (10) further comprises at least one reservoir (13), in which at least one detection reagent comprising specific detection antibodies (55a) with a label (55b) for binding to the respective analytes and / or comprising competitive analytes with a label for binding to optionally still free specific capture antibodies (5a, 5ab, 5c) and / or at least one washing solution and / or at least one readout reagent are arranged upstream.Microfluidic device (10), in particular cartridge (100), according to one of the preceding claims, further comprising at least one electrochemical and / or optical readout unit (9), in particular a camera (9), for detecting the respective n analytes bound to the bead-coupled specific capture antibodies (5a, 5b, 5c), and an evaluation unit configured to determine a concentration of the n analytes by means of an evaluation algorithm, in particular based on artificial intelligence, and in particular further comprising at least one temperature control device (7) and / or a display unit.Microfluidic system comprising a microfluidic device (10) according to one of the preceding claims, embodied as a cartridge (100), and an analysis device comprising at least one electrochemical and / or optical readout unit (9), in particular a camera (9), for detecting the respective n analytes bound to the specific bead-coupled capture antibodies (5a, 5b, 5c) and an evaluation unit configured to determine the concentration of the n analytes by means of an evaluation algorithm, in particular based on artificial intelligence, and in particular further comprising at least one temperature control device (7) and / or a display unit, wherein the analysis device is configured to process the cartridge (100) and / or to read results therefrom.Microfluidic system according to claim 8, wherein the readout unit (9) is movably mounted so that it is movable towards each retaining chamber (14a, 14b, 14c) during readout or wherein the readout unit can read all retaining chambers (14a, 14b, 14c) simultaneously.Method (50) for the multiplexed determination of a concentration of n analytes, wherein n is a natural number >1, in a sample, in particular a urine sample, by means of a microfluidic device (10) according to one of claims 1 - 7 or by means of a microfluidic system according to one of claims 8 or 9, with the following steps a) providing the sample and inputting a sample volume into the microfluidic device (10), b) guiding the sample into the, in particular temperature-controlled, reaction chamber (3) and binding the first analyte to the first specific capture antibody (5a) coupled to the first bead (2a) with formation of a first bead antibody analyte complex and binding the at least one nth analyte to the nth specific capture antibody (5b, 5c) coupled to the nth bead (2b, 2c) to form an nth bead antibody analyte complex c) conducting the sample with the first and the at least one nth bead antibody analyte complex into the first retaining chamber and size selective retention of the at least one first bead antibody analyte complex on the first retaining element (14a), and passing the (n-1)th retaining element (14a) through the at least one nth bead antibody analyte complex. d) conducting the sample with the at least one nth bead antibody analyte complex into the nth retaining chamber (14b, 14c) and size-selective retention of the at least one nth bead antibody analyte complex on the nth retention element (14b, 14c) e) addition of a specific detection reagent into the microfluidic device (10), in particular from a reservoir (13) thereof, wherein the specific detection reagent comprises specific detection antibodies (55a) with a label (55b), in particular an enzyme, and binding of these to the respective bead antibody analyte complexes and / or wherein the specific detection reagent comprises competitive analytes with a label, in particular an enzyme, and binding of these to optionally still free specific capture antibodies (5a, 5ab, 5c). f) addition of a readout reagent, in particular from a reservoir (13) of the microfluidic device (10), and detecting a reaction catalyzed by the label (55b) of the detection antibodies (55a) and / or by the label of the competitive analytes, in particular a color and / or radiation intensity, by means of a readout unit (9), in particular a camera (9). g) determining the concentration of the n analytes on the basis of the detected reaction, in particular the color and / or radiation intensity, and in particular comparing these with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations takes place in the evaluation unit with the aid of an evaluation algorithm, in particular based on an artificial intelligence.Method according to claim 10, wherein in step b) the sample is allowed to stand and / or the sample is moved gently in the reaction chamber (3) for a predetermined time, in particular for 1-15 min.Method according to one of claims 10 or 11, wherein steps c) and d) are repeated by pumping the sample in a circuit, in particular with a defined dwell time, in particular for 0.1-1 min, after each pump cycle.Method according to one of Claims 10-12, wherein in steps b) and / or c) and d) at least a temperature control of the reaction chamber (3) and / or of the retaining chambers takes place in regions, in particular at 21-40°C.Method according to any one of claims 10-13, wherein the n analytes are hormones, in particular luteinizing hormone (LH) and / or follicle stimulating hormone (FSH) and / or tyreotropin (TSH) and / or human chorionic gonadotropin (hCG) and / or anti-Muller hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.Method according to one of claims 10 - 14, wherein after step g) in a step h) the respective concentration of the n analytes is output and / or a recommendation for action and / or an item of information, in particular a phase and / or a day in the female cycle and / or a fatiguable window of a subject is calculated and output, in particular on a display unit and / or in an associated app.

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