Device and method for analysing a hormone concentration

Abstract

Proposed is a microfluidic device (10) for the multiplex determination of a concentration of n analytes in a sample, the device comprising: a reaction chamber (3) which has a number n of different beads (2a, 2b, 2c) of successively decreasing diameter, wherein beads of different diameters (2a, 2b, 2c) are coupled to different specific capture antibodies (5a, 5b, 5c); and a number n of different microfluidically interconnected retention chambers each having a first retention element (14a, 14b, 14c), wherein the pores of the first retention elements (14a) have different diameters, and wherein the diameter of the at least one first bead (2a) is such that said at least one first bead can be retained on the first retention element (14a), and wherein the at least one n-th bead (2b, 2c) has a diameter smaller than the diameter of the pores of the first retention element (14a) but can nevertheless be retained on the n-th retention element (14b, 14c).

Description

[0001] Description

[0002] title

[0003] Device and method for analyzing a hormone concentration

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

[0005] State of the art

[0006] According to theory, the female cycle is divided into menstruation, follicular phase, ovulation, and luteal phase. The relative changes in the relevant variables, namely the hormones, herald the different phases. The concentration ranges of the hormones in the blood and urine are known and serve as a basis for gynecologists to diagnose imbalances.

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

[0008] Basal thermometers and cycle apps, sometimes combined, provide indirect parameters about the status of a woman's cycle. Basal body temperature only rises shortly before ovulation, making it impossible to determine the so-called fertile window (approximately four days before). This can result in unwanted pregnancies or a reduced chance of becoming pregnant.

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

[0010] So-called lab-on-a-chip systems, for example, comprise two main components. The first is a test carrier, for example in the form of a cartridge, which contains structures and mechanisms for manipulating a sample, particularly passive components such as channels or reaction chambers, or active components such as valves, pumps, or mixers. The second main component is a processing unit for controlling the microfluidic processes in the cartridge, such as actuating the valves or pumps, as well as for detecting sample components before, during, and / or after processing.

[0011] WO 2014 / 151456 A2 discloses a multi-chamber measurement system for antibody-based determination of various hormones.

[0012] EP 0751782 A1 discloses a test for determining the hormones of the female cycle in the blood.

[0013] Disclosure of the invention

[0014] According to the invention, a microfluidic device, in particular a cartridge, for diagnosis at the point of care or at home, a system comprising the microfluidic device, in particular cartridge, and a method for operating the same are provided, having the features of the independent patent claims.

[0015] This is based in particular on the fact that the microfluidic device, in particular a cartridge, is configured for the multiplexed determination of a concentration of n analytes, where n is a natural number greater than 1, thus at least two analytes in a sample, in particular a biological sample such as a urine sample or a blood sample. The microfluidic device, in particular the cartridge, comprises a reaction chamber having a number n of different beads with a successively decreasing diameter, each bead coupled to a specific capture antibody.

[0016] The first bead coupled to the first specific capture antibody has a first diameter, and the at least one further n-th bead coupled to an n-th specific capture antibody (5a) has a smaller diameter than the (n - 1)th bead. The symbol " represents a minus. If, for example, two analytes are determined in the sample, then n = 2, and the specific, capture antibody-coupled second beads 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, capture antibody-coupled third beads have a smaller diameter than the (3 - 1)th bead, i.e., the second bead, and so on.

[0017] A bead can be understood as a microparticle that 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.

[0018] 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 pm to 1 cm and is made, for example, of a polycarbonate (PC), a polypropylene (PP), polystyrene and / or a cycloolefin copolymer (COP) in order to minimize reactions of the analytes on non-coated surfaces.

[0019] Furthermore, the microfluidic device, in particular the cartridge, comprises a number n of retention chambers, each with a retention element. The first retention chamber is microfluidically connected to the at least one nth retention chamber. The retention element is, for example, a microfilter or a microsieve. The diameter of the pores of the first and the at least one nth retention element is, for example, in a range from 25 nm to several hundred nm. The retention 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 non-magnetic metals.

[0020] The retention chambers have dimensions of 0.5 to 5 cm in diameter and heights of 200 pm to 0.5 cm and are made of PC, COP or a coated polymer such as bovine serum albumin (BSA) to avoid reactions of the analytes on non-coated surfaces.

[0021] The retention chambers have, for example, an inlet located laterally above the retention element in the flow direction and not directly above the surface of the retention element fixed therein, and an outlet located, for example, below the filter in the flow direction, particularly on the opposite side of the inlet. This can be realized, for example, over two levels.

[0022] At least on an upper side, which is located above the retaining element in the flow direction, the retention chamber for selection advantageously has a transparent, translucent material, for example a transparent PC, COP and / or polystyrene (PS).

[0023] 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. For example, if n is two, the pores of the second retaining element have a smaller diameter than the (2 - 1)th retaining element, i.e., the first retaining element. For example, if n is three, the pores of the third retaining element have a smaller diameter than the (3 - 1)th retaining element, i.e., the second retaining element.

[0024] Furthermore, the diameter of the at least one first bead is larger than the diameter of the pores of the first retaining element, so that the at least one first bead is retained on the first retaining element. Furthermore, the diameter of the at least one n-th 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 n-th retaining element, so that the at least one n-th bead is retained on the n-th retaining element.

[0025] The advantage here is that a very precise measurement of the concentration of the analytes, especially different types of analytes, such as different types of hormones, especially peptide hormones and steroid hormones, in a sample is possible at the point of care or conveniently at home for an on-site diagnosis.

[0026] The respective analytes can be detected specifically and sensitively, allowing their concentrations to be determined with the same accuracy as in a laboratory analysis by a physician or in a central laboratory. The measurement is performed in multiplex, as the n analytes can be captured simultaneously, specifically read out simultaneously, and the analyte concentrations can be determined in parallel and using the same method. This saves time until the result is obtained, as well as practical labor, work steps, containers (especially reservoirs), and the reagents themselves.

[0027] Another particularly advantageous feature is that the analyte concentration can be determined at the point of care or at home, making it very simple, convenient, and user-friendly. A urine or saliva sample, for example, is used for this purpose. The advantage here is that it can be obtained painlessly and without anxiety, using a simple, non-invasive method. Alternatively, a blood sample, for example, can also be used.

[0028] Compared to a lateral flow format, the microfluidic environment increases sensitivity, specificity, and the degree of multiplexing, while also saving reagents compared to individual tests. Furthermore, temperature control, dilution, and washing steps are possible.

[0029] Another advantage is that the sample can be prepared for analysis, for example by automated dilution on the microfluidic device, in particular the cartridge.

[0030] The reaction chamber and the retention chambers are connected to each other via a fluidic network, whereby a sample is first fed into the reaction chamber and then into the respective retention chambers with the retention elements, starting with the first retention chamber with the first retention element with the largest pore diameter and finally into the next retention chamber with the retention element with the next smallest pore diameter. In a preferred embodiment, the retention chamber with the retention element with the smallest pore diameter is in turn fluidically connected to the reaction chamber, so that the sample can be circulated. In an alternative embodiment, the sample is discharged into a waste container after passing through the retention chambers.Fluids, in particular reagents, samples and buffers, are transported through the microfluidic network of the microfluidic device or cartridge by pressure, for example by means of membrane pumps and / or by gravity and / or by means of charge differences.

[0031] The reaction chamber and / or the retention chambers can, for example, be temperature-controlled, in particular heated, at least in some areas, so that optimal binding conditions for the binding of the analytes to the respective capture antibodies are present in the reaction chamber and / or so that optimal binding conditions for the binding of detection antibodies to a further binding site of the analyte and / or for the binding of competitive analytes to the capture antibodies are present in the retention chambers.

[0032] The microfluidic device according to the invention is designed, for example, as a lab-on-chip cartridge, hereinafter referred to as a cartridge. The cartridge includes interfaces to another unit, for example, an analytical device, and may include additional components.

[0033] Further advantageous embodiments of the microfluidic device emerge from the subclaims.

[0034] In a further advantageous embodiment, the antibody-coupled beads comprise a magnetic material, in particular gold, iron, and / or platinum, and the retention chambers are at least partially magnetic in a region located below the retention element in the flow direction. Alternatively, a magnet is arranged below the retention chamber in the flow direction. The advantage here is that the magnetic antibody-coupled beads are thus drawn toward the magnetically designed region of the retention chamber or toward the magnet below the retention chamber and, if the diameter of the bead is larger than the pore diameter of the retention element, collect at one point on the retention element.This results in a concentration of the signal intensity in a smaller space, so that during readout, a large luminous or colored point can be detected instead of a signal intensity distributed over the entire surface of the retention element in the form of individual small luminous points. This increases sensitivity. In addition, the readout unit, in particular the camera, can be constructed more simply. If the diameter of the bead is smaller than the pore diameter of the retention element, the antibody-coupled bead is pulled through it. The magnet also enables 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 antibodies in an embodiment described below, in which the sample or analyte is transported several times through the retention chambers in a circuit, because a spatial concentration takes place.

[0035] 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 tyrotropin (TSH) and / or the human chorionic gonadotropin (hCG) and / or the anti-Müllerian hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.

[0036] In an advantageous embodiment, the capture antibodies coupled to the beads are at least specific for LH, FSH, hCG, estrogen and progesterone.

[0037] These hormones reflect a woman's cycle status / fertility status particularly well, so that, for example, the entire fertile window of a test subject can be determined very precisely. Further advantages of analyzing these hormones are listed later in the application. Furthermore, in an advantageous embodiment, the microfluidic device or cartridge comprises at least one reservoir in which at least one detection reagent comprising specific detection antibodies that bind to the respective analytes and / or competitive analytes for binding to any still free specific capture antibodies is stored. The detection antibodies and / or the competitive analytes carry a label for selection. Additionally or alternatively, at least one washing solution and / or at least one selection reagent and / or at least one buffer for dilution is stored in the at least one reservoir.By means of a microfluidic connection from the at least one reservoir to a first retention chamber and / or to the reaction chamber, the upstream reagents can be directed thereto.

[0038] In one embodiment, the microfluidic device comprises at least one electrochemical and / or optical readout unit for detecting the respective analytes bound to the bead-coupled specific capture antibodies.

[0039] 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.

[0040] An advantage of using a readout unit compared to lateral flow tests without a readout unit is, for example, that the sensitivity is increased.

[0041] The readout unit is, for example, movably mounted so that it can be moved towards the retention chambers during reading. Alternatively, the readout unit is configured to read all retention chambers simultaneously. Further alternatively, a readout unit is provided for each retention chamber. At least the top side, i.e. the side of the retention chamber which is located above the retention element as seen in the flow direction, 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 retention element can be read out. Furthermore, the microfluidic device comprises an evaluation unit configured to determine the concentration of the n analytes using 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 a basis for evaluation. In this way, for example, deviations from natural concentrations of the respective analytes can be detected and / or natural or unnatural fluctuations in the analytes can be detected, for example natural or unnatural fluctuations in hormones. The advantage here is that, if a user has sufficient data, the software can, for example, determine deviations from the individual hormone level over the cycle. Furthermore, it is advantageous that an overall picture of all determined concentrations of the analytes is determined in this way, in contrast to the state of the art where this is often only possible selectively for individual analytes.Based on this, a recommendation for action and / or information is advantageously issued to the user.

[0042] The advantage is that the use of artificial intelligence allows for quantification of the analytes and enables evaluation with customer-oriented recommendations for action and / or the output of information, instead of a test strip result that has to be interpreted by the user in a readout window.

[0043] In particular, the microfluidic device further comprises a display unit, for example a display, on which the issued recommended action and / or information and / or the determined analyte concentrations are displayed. Additionally or alternatively, the recommended action and / or information and / or the determined analyte concentrations are transmitted to an associated app and displayed to the user via this app.

[0044] In an advantageous embodiment, the microfluidic device comprises at least one temperature control device for controlling the temperature, in particular heating, of the reaction chamber and / or the retention 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 the size of a smartphone, which can be conveniently transported, for example, in a handbag.

[0045] Furthermore, according to the invention, a microfluidic system is provided, comprising a microfluidic device according to the invention, which is designed as a cartridge, and an analysis device. The analysis 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 analysis device is designed to process the cartridge and / or read out results.

[0046] For example, the reading unit is mounted so that it can be moved toward each retention chamber during the reading process. Alternatively, the reading unit is designed to read all retention chambers simultaneously.

[0047] Further statements regarding the readout unit already mentioned above apply here accordingly.

[0048] The evaluation unit is preferably programmed accordingly together with a processor in the analyzer. The further comments regarding the evaluation unit mentioned above apply here accordingly.

[0049] Furthermore, the analyzer comprises, in particular, a display unit, to which the above-mentioned statements also apply, as well as with regard to the app mentioned therein. Furthermore, the analyzer comprises an optional temperature control device, to which the above-mentioned statements also apply. Furthermore, the analyzer comprises, for example, a magnet.

[0050] The cartridge, for example, is designed as a disposable part, while the analyzer is a multi-use unit. The analyzer is about the size of a smartphone, for example, which can be easily carried in a handbag. The cartridge is smaller than the analyzer, about the size of a matchbox, for example.

[0051] The analysis device (processing unit) and the cartridge can be designed, for example, as described in DE102016222072A1 or DE102016222075A1 and the cartridge can be processed accordingly.

[0052] In a further embodiment of the system, both the microfluidic device, in particular the cartridge, and the analysis device comprise an optical and / or electrochemical readout unit and / or an evaluation unit and / or a temperature control device and / or a magnet. The readout and / or evaluation units can complement each other, for example.

[0053] The invention further relates to a method for the multiplexed determination of a concentration of n analytes, where n is a natural number >1, in a sample, in particular in a biological sample such as a urine sample by means of the microfluidic device or by means of the microfluidic system with the following steps: a) Providing the sample and introducing a particularly defined sample volume into the microfluidic device.

[0054] The sample, for example, a urine sample, is collected in a container, and a particularly defined sample volume is introduced into the microfluidic device, in particular a cartridge, by a user, for example, using a pipette or other device, for example, via a microfluidic inlet. If the sample concentration is too high, it is diluted, for example, automatically, so that subsequent reading can be carried out reliably and without interference.

[0055] A particular advantage is that the sample can be collected and analyzed at home or at the point of care, saving a significant amount of time. Another advantage is that if the sample is urine or saliva, it can be obtained painlessly and without anxiety, using a simple, non-invasive method. Alternatively, a blood sample can be used, for example.

[0056] The input of a defined sample volume is particularly advantageous, as this enables standardization. b) Passing the sample into a reaction chamber (3), in particular a temperature-controlled one, and binding of 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 of 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. The advantage here is that only the appropriate analyte can bind to the specific capture antibody, thus allowing a very specific and accurate result to be obtained. Furthermore, the analytes can be determined in multiplex.

[0057] Advantageously, in step b), the sample is left to rest for a predetermined time, in particular for 1-15 minutes. Alternatively, the sample is gently agitated in the reaction chamber. An incubation period in which the sample remains at rest or gently agitated in the reaction chamber promotes the binding of the analytes to the respective specific bead-coupled capture antibodies, so that a very precise determination of the concentration of the analytes in the sample can be carried out. c) Passing 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) passing the sample with the at least one n-th bead-antibody-analyte complex into the n-th retention chamber and size-selective retention of the at least one n-th bead-antibody-analyte complex on the n-th retention element.

[0058] The advantage of steps c) and d) is that this method allows for a very good separation of the various analytes in the sample, which ultimately allows for a very precise determination of the analyte concentration. Another particularly advantageous feature is that this type of separation of the analytes in a multiplex allows the same readout unit to be used for subsequent readout of all n analytes.

[0059] Advantageously, steps c) and d) are repeated by pumping the sample in a circuit, in particular with a defined standing time, in particular for 0.1 - 1 min, after each pumping cycle.

[0060] This advantageously increases the chances of binding of still unbound analytes to the respective specific bead-coupled capture antibodies.

[0061] Advantageously, after step d), a washing step is carried out by adding a washing reagent and circulating it through the reaction chamber and the retention chambers. The washing reagent is advantageously stored upstream, in particular in a reservoir of the microfluidic device, in particular the cartridge. e) Supplying a specific detection reagent, in particular from a reservoir, and circulating it through the retention chambers, wherein the specific detection reagent comprises specific detection antibodies with a label, in particular an enzyme, and binds these to the respective analyte of the bead-antibody-analyte complexes and / or wherein the specific detection reagent comprises competitive analytes with a label, in particular an enzyme, and binds these to any still free specific capture antibodies. Competitive analytes are artificial analytes for the analytes to be detected.

[0062] If the specific bead-coupled capture antibodies already carry a label for readout, step e) is not necessary. All detection antibodies and / or competitive analytes advantageously carry the same label for readout. Optionally, one or more detection antibodies can also be present without a label or, for example, with a biotin label. In this case, a species-specific secondary antibody is required, for example, which binds the detection antibody and carries the label for readout or, in the case of the biotin label, a streptavidin-enzyme complex that binds to the biotin and carries the label for readout. An enzyme, for example, horseradish peroxidase (HRP) or alkaline phosphatase (AP), serves as the label for readout.

[0063] Advantageously, after adding the detection reagent, at least one further washing step analogous to the washing step already described is performed to wash out unbound detection antibodies and / or unbound competitive analytes. The washing reagent is advantageously stored upstream, in particular in a reservoir of the microfluidic device and in particular the cartridge. f) Adding a readout reagent, in particular from a reservoir of the microfluidic device or the cartridge.The readout reagent for detecting the labeled detection antibody and / or the competitive analyte is, for example, a substrate such as TMB (3, 3', 5,5'-tetramethylbenzidine) and causes the label, for example the enzyme of the detection antibody or the competitive analyte, to convert the substrate, causing a color reaction and / or a chemiluminescent reaction in the form of an emission of electromagnetic radiation in the ultraviolet and visible light range, and / or a fluorescent reaction in the form of an emission of electromagnetic radiation.

[0064] For example, a reaction catalyzed by horseradish peroxidase can be read colorimetrically, chemifluorescently, or chemiluminescently. A reaction induced by alkaline phosphatase can be read colorimetrically or chemiluminescently.

[0065] A readout unit, particularly a camera, records the intensity of the color reaction or the density of the generated dye and / or the emitted electromagnetic radiation. With competitive analytes, if a large amount of analyte is present in the sample, few competitive analytes will be bound, since the binding sites of many specific capture antibodies, to which the competitive analytes can then bind, are already occupied by the analyte. Thus, a low signal is detected during readout. If there is little analyte in the sample, the binding sites of many specific capture antibodies remain free, to which the competitive analytes can then bind. A high signal is then detected during readout.

[0066] It is advantageous that for the detection of different analyte types, where in one embodiment at least one analyte type is detectable using labeled detection antibodies and at least one analyte type is detectable using competitive analytes, and thus the intermediate steps are different, the readout step can still be carried out by the same readout system, adding the same readout reagent, and detectable using the same readout unit. This allows for a high degree of analyte multiplexing. Furthermore, costs are saved because only one readout system, for example, a camera with a filter, is required.

[0067] Following step f), at least one washing step analogous to the previously described washing steps is advantageous. g) Determining the concentration of the n analytes based on the detected reaction, in particular the color and / or radiation intensity, and in particular comparing this with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations is carried out in the evaluation unit using an evaluation algorithm, in particular based on artificial intelligence.

[0068] If necessary, it is advantageous to prepare the sample for the process before step a) by, depending on the sample, separating interfering substances and particles, for example, using a filter, or, for example, in the case of urine samples, separating suspended matter, for example, using a centrifuge, or, in the case of blood samples, performing plasma separation. Furthermore, in one embodiment, it is advantageous if, in steps b) and / or c) and / or d), at least some of the reaction chamber and / or the retention chambers are heated, in particular at 21-40°C. The heating or cooling takes place across the entire chamber or in individual areas thereof.

[0069] The advantage here is that in this way optimal local binding conditions are present in terms of temperature for the binding of the analytes to the respective specific bead-coupled capture antibodies and, when the sample is circulated, also for the binding of the detection antibodies to the analytes and / or the competitive analytes to the specific capture antibodies.

[0070] 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 tyrotropin (TSH) and / or the human chorionic gonadotropin (hCG) and / or the anti-Müllerian hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.

[0071] The advantage of determining the concentrations of these hormones is that they are relevant hormones of the female cycle. Measuring their concentrations allows conclusions to be drawn about this, such as the day of the female cycle or the fertile days. Furthermore, natural or unhealthy hormone concentrations and fluctuations can be determined, which allow conclusions to be drawn about possible illnesses or phases, such as menopause or favorable training phases for athletes.

[0072] 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.

[0073] These hormones reflect a woman's cycle status particularly well, so that they can be used to determine, for example, a test subject's entire fertile window very precisely. In an advantageous embodiment, after step g), in a step h), the respective concentration of the n analytes is output and / or, based on 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 test subject's fertile window, is calculated and output. The recommendation for action and / or information is displayed in particular on a display unit of the microfluidic device or 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 via this app.The advantage here is that, instead of having to interpret a strip in the readout window of existing lateral flow tests, a test subject receives a concentration of the analytes and / or information and / or a recommendation for action on their smartphone or the display unit, especially a screen. This avoids uncertainty on the part of the test subjects, as well as unwanted pregnancies or unnecessary intake of active substances, and increases the chances of becoming pregnant if they wish to conceive.

[0074] Short description of the drawing

[0075] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows:

[0076] Fig. 1 : the schematic representation of a microfluidic device according to the invention in a first embodiment before the start of the process,

[0077] Fig. 2: the schematic representation of the microfluidic device according to the invention according to Fig. 1 during the process implementation,

[0078] Fig. 3: the schematic representation of the inventive

[0079] Device according to Figure 2, which is designed in the form of a cartridge, and

[0080] Fig. 4: The schematic representation of an embodiment of the method according to the invention. Embodiments of the invention

[0081] Figure 1 shows a first embodiment of the microfluidic device 10 according to the invention. This can be designed as a cartridge 100. Using the microfluidic device 10 in Figure 1, the concentrations of three analytes, i.e., n=3, in a sample can be determined in multiplex. This is described below by way of example for all analytes using 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 2a coupled to a first specific capture antibody 5a, a second bead 2b coupled to a second specific capture antibody 5b, and a third bead 2c coupled to a third specific capture antibody 5c are disposed in a mixture.

[0082] The first bead 2a has a diameter that is larger than the diameter of the second bead 2b. The diameter of the second bead 2b, in turn, is larger than the diameter of the third bead 2c.

[0083] The first specific capture antibody 5a binds specifically to LH, the second specific capture antibody 5b binds specifically to FSH and the third capture antibody 5c binds specifically to progesterone.

[0084] The reaction chamber, for example, has a length and a width of 0.5 cm to 5 cm each and a height of 300 pm to 1 cm

[0085] The reaction chamber 3 is microfluidically connected to a first retention chamber with an integrated first retention element 14a. The first retention chamber is microfluidically connected to a second retention chamber with a second retention element 14b, and the second retention chamber is microfluidically connected to a third retention chamber with a third retention element 14c. Figure 1 shows only the retention elements 14a, 14b, 14c, not the retention chambers. The retention elements 14a, 14b, 14c are, for example, microfilters or microsieves made of a silica-based material. The pores of the first retention element 14a have a larger diameter than the pores of the second retention element 14b, and the pores of the second retention element 14b have a larger diameter than the pores of the third retention element 14c.

[0086] The diameter of the pores of the first, second and third retaining elements 14a, 14b, 14c is, for example, in a range from 25 nm to several hundred nm. The retention chambers have, for example, dimensions of 0.5 cm to 5 cm in diameter and heights of 200 pm to 0.5 cm and are made, for example, of PC, COP and / or PS.

[0087] Furthermore, the diameter of the first bead 2a is larger than the diameter of the pores of the first retaining element 14a, so that the first bead 2a is retained on the first retaining element. Furthermore, the diameter of the second bead 2b is smaller than the diameter of the pores of the first retaining element 14a, but larger than the diameter of the pores of the second retaining element 14b, so that the bead 2b is retained on the second retaining element 14b. Furthermore, the diameter of the third bead 2c is smaller than the diameter of the pores of the second retaining element 14b, but larger than the diameter of the pores of the third retaining element 14c, so that the third bead 14c is retained on the third retaining element.

[0088] The third retention chamber is microfluidically connected to the reaction chamber 3 so that a fluid can be circulated through the microfluidic device 10.

[0089] The sum of all microfluidic connections or channels forms a microfluidic network 19.

[0090] 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 Figure 1, and / or at least one washing solution and / or at least one readout reagent and / or at least one buffer for dilution is stored. Advantageously, the microfluidic device 10 has multiple reservoirs 13. Furthermore, the microfluidic device has, for example, a waste container (not shown in Figure 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 retention chambers 14a, 14b, 14c and / or the reaction chamber 3 via the fluidic network 19. The microfluidic channels 19 are opened and closed via valves 11. The flow direction through the microfluidic device is shown in Figure 1 by small arrows on the microfluidic channels.

[0091] The retention chambers have, for example, an inlet located laterally above the respective retention element 14a, 14b, 14c in the flow direction and not directly above the surface of the latter, and an outlet located, for example, below the filter in the flow direction, in particular on the opposite side of the inlet. This can be realized, for example, over two levels. This is not shown in Figure 1.

[0092] At least on an upper side, which is located above the retention element in the flow direction, the retention chamber has a transparent material for selection, for example a transparent PC, COP, PS and / or glass.

[0093] The microfluidic device 10 comprises a microfluidic inlet 1, shown as an arrow in Figure 1, via which a sample, for example a urine sample, can be introduced into the device 10.

[0094] Furthermore, at least one region of the reaction chamber 3 and / or the retention chambers 14a, 14b, 14c can be temperature-controlled. For this purpose, the microfluidic device 10, or if it is configured, for example, as a cartridge 100, comprises an analysis device (not shown in the figures), a temperature control device 7 (shown only symbolically in Figure 1), for example, a heater or a Peltier element. If the microfluidic device 10 is configured as a cartridge 100, the cartridge 100 comprises, for example, interfaces to the temperature control device 7 arranged on the analysis device.

[0095] Advantageously, the antibody-coupled beads 2a, 2b, 2c comprise a magnetic material, in particular gold, iron, and / or platinum, which can additionally be functionalized, for example, with dextran derivatives such as carboxymethyldextran (CMD) and / or aminodextran (AMD) for improved biofunctionality. The retention chambers are, for example, at least partially magnetic in a region located below the retention element 14a, 14b, 14c in the flow direction. Alternatively, a magnet 8 is arranged below the corresponding retention chamber in the flow direction and / or in an analysis device in a position located below the retention chamber. This is shown only symbolically in Figure 1. The flow direction through the microfluidic device 10 is indicated by small arrows on the microfluidic channels of the microfluidic network 19.

[0096] Furthermore, Figure 1 also symbolically depicts a readout unit 9 in the form of a camera 9, by means of which the hormones bound to the specific capture antibodies 5a, 5b, 5c on the retention elements 14a, 14b, 14c can subsequently be read out. The camera 9 is movably mounted, as indicated by the arrows 9b. In Figure 1, the readout area 9a of the camera 9 is intended to illustrate that all retention elements 14a, 14b, 14c are read out by one camera 9. The readout unit 9 is, for example, a part of the microfluidic device 10 itself or, if the microfluidic device 10 is designed, for example, as a cartridge 100, the cartridge 100 comprises an interface to a readout unit 9 located in an analysis device.

[0097] Figure 2 shows the microfluidic device according to Figure 1 during the process. The beads 2a, 2b, 2c coupled with a specific capture antibody 5a, 5b, 5c have already been 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 14b, and third bead-antibody-progesterone complexes are retained on the third retaining element 14c. For readout by the readout unit, which is designed as a camera 9 in Figure 1, the readout unit is movably mounted, for example, so that it can be moved toward the retention chambers 14a, 14b, 14c during readout. This is shown as an example by the arrows 9b to the right and left of camera 9.

[0098] Alternatively, the readout unit is configured to read all retention chambers 14a, 14b, 14c simultaneously. Alternatively, a separate readout unit is provided for each retention chamber 14a, 14b, 14c. At least the top side, i.e., the side of the retention chamber located above the retention element 14a, 14b, 14c as seen in the flow direction, is made of a transparent material, for example, a transparent PC, COP, glass, and / or PS, so that the coupled analytes retained on the retention element can be read out. In Figure 2, the readout area 9a of the camera 9 is intended to illustrate that all retention elements 14a, 14b, 14c are read out by one camera 9; this can, but does not have to, be the same camera.

[0099] 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 analysis device. The evaluation unit is configured to determine a concentration of n analytes.

[0100] 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 analysis device.

[0101] In addition, the microfluidic device, in particular designed as a cartridge, or the analysis device comprises, for example, at least one pump, in particular a membrane pump.

[0102] 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 in the form of a one-piece device, or alternatively, the microfluidic device 10 according to Figure 2 is designed as a cartridge 100 - as shown in Figure 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 analysis device (not shown). The cartridge then has interfaces to the analysis device (not shown in the figures). The cartridge 100 can have further components (not shown).

[0103] Figure 4 shows a flow diagram of an embodiment of the method 50 according to the invention for the multiplexed determination of a concentration of three analytes present in a sample at the point of care or at home.

[0104] Representing other or additional analytes, the method according to the invention is described below using 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, from a female test subject, for example, is introduced into the microfluidic device 10 or the cartridge via the fluidic inlet 1, for example by means of a pipette.

[0105] In a step b), the sample is introduced into the reaction chamber 3, which is in particular tempered at 21 -40 °C, in which bead-coupled specific capture antibodies 5a, 5b, 5c are stored.

[0106] For a predetermined time, in particular for 1-15 minutes, the sample is left to rest or gently moved in the reaction chamber 3.

[0107] The first specific capture antibody 5a, coupled to the first bead 2a, binds specifically to LH, forming a first bead-antibody-LH complex. The second specific capture antibody 5b, coupled to the second bead 2b, binds specifically to FSH, forming a second bead-antibody-FSH complex, and the third specific capture antibody 5c, coupled to the third bead 2c, binds specifically to progesterone, forming a third bead-antibody-progesterone complex. For simplicity, these are referred to below as the first complex, second complex, and third complex.

[0108] In a further step c), the sample containing the at least one first, second, and third complex is passed into the first retention chamber with the first retention element 14. The first complex is retained on the first retention element 14a due to its size, based on the diameter of the first bead 2a. The second and third complexes can pass through the first retention element 14a.

[0109] In a further step d), the sample containing the second and third complexes is passed into the second retention chamber with the second retention element 14b. The second complex is retained there due to its size, due to the diameter of the second bead 2b. The third complex passes through the second retention element 14b.

[0110] The sample with the third complex is then passed into the third retention chamber with the third retention element 14c. The third complex is retained on this chamber due to its size, due to the diameter of the third bead 2c. Thus, the LH from the sample is located 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.

[0111] Steps c) and d) are repeated by circulating the sample, particularly with a defined dwell time of 0.1-1 min after each pumping cycle. In steps c) and d), the first, second, and third retention chambers are advantageously tempered, particularly to 21-40°C.

[0112] This is followed by a washing step in which a washing reagent is added from a reservoir 13. This is flushed circularly through the reaction chamber and the retention chambers.

[0113] Subsequently, in a step e), a specific detection reagent 55, in particular one stored upstream in a reservoir 13, is added, wherein the specific detection reagent 55 comprises specific detection antibodies 55a with a label 55b and / or competitive analytes with a label. The specific detection antibodies 55a bind specifically to the peptide hormones LH and FSH in the respective bead-antibody-hormone complex. Competitive analytes to progesterone bind to the possibly still free capture antibodies (5a, 5ab, 5c), which are specific for progesterone. Advantageously, all detection antibodies 55a and / or competitive analytes carry the same label 55b for later selection. An enzyme, for example HRP, serves as the label.

[0114] Advantageously, after the addition of the detection reagent 55, at least one further washing step is carried out, which is carried out analogously to the washing step described above.

[0115] Subsequently, in a step f), a readout reagent is added, in particular from a reservoir 13 of the microfluidic device 10 or the cartridge 100. The readout reagent for detecting the detection antibody 55a with the label 55b and / or the competitive analyte with the label is, for example, a substrate such as TMB (3,3',5,5'-tetramethylbenzidine) and causes the label 55b of the detection antibody 55a and / or the competitive analyte to react with the substrate, thereby causing a color reaction and / or a chemiluminescent reaction in the form of an emission of electromagnetic radiation in the ultraviolet and visible light range, and / or a fluorescent reaction in the form of an emission of electromagnetic radiation. The reaction catalyzed by HRP can be read out, for example, colorimetrically, chemifluorescently or chemiluminescently.

[0116] A readout unit 9, in particular a camera 9, detects the intensity of the color reaction or the density of the generated dye and / or the emitted electromagnetic radiation.

[0117] Following step g), at least one washing step analogous to the washing steps described previously is advantageous.

[0118] In a subsequent step g), the concentrations of the hormones LH, FSH, and progesterone are determined based on the color and / or radiation intensity detected by the reading unit 9 using software comprising an evaluation algorithm, in particular based on artificial intelligence. Furthermore, the evaluation unit advantageously compares the determined hormone concentrations in the sample with known concentrations and evaluates and classifies them.

[0119] In step h), for example, the respective concentrations of the hormones LH, FSH, and progesterone are output. 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 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 illnesses or phases, such as menopause or favorable training phases for athletes.

Claims

Claims 1 . Microfluidic device (10), in particular a cartridge (100) for a diagnosis at the point of care or at home, for the multiplexed 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 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 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 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 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 (n - 1)th retention element (14a), but larger than the diameter of the pores of the nth 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) 2. Microfluidic device (10), in particular cartridge (100) according to claim 1, wherein the specific capture antibodies (5a, 5b, 5c) are further each coupled to a label, in particular an enzyme, for selection.

3. 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 retention chambers are at least partially magnetic in a region which is located below the respective retention element (14a, 14b, 14c) in the flow direction, or wherein a magnet (8) is arranged below the respective retention chamber in the flow direction.

4. 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 tyrotropin (TSH) and / or the human chorionic gonadotropin (hCG) and / or the anti-Müllerian hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.

5. 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 luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), estrogen and progesterone.

6. 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 possibly still free specific capture antibodies (5a, 5ab, 5c) and / or at least one washing solution and / or at least one reading reagent are preceded.

7. 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.

8. A microfluidic system comprising a microfluidic device (10) according to any one of the preceding claims, designed 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 designed to process the cartridge (100) and / or read out results from it.

9. Microfluidic system according to claim 8, wherein the readout unit (9) is movably mounted so that it can be moved towards each retention chamber (14a, 14b, 14c) during the readout or wherein the readout unit can read all retention chambers (14a, 14b, 14c) simultaneously.

10. Method (50) for the multiplexed determination of a concentration of n analytes, where 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 introducing a sample volume into the microfluidic device (10), b) Leading the sample into the, in particular temperature-controlled, reaction chamber (3) and binding of the first analyte to the first specific capture antibody (5a) coupled to the first bead (2a) to form a first bead-antibody-analyte complex and binding of the at least one n-th analyte to the n-th specific capture antibody (5b, 5c) coupled to the n-th bead (2b, 2c) to form an n-th bead-antibody-analyte complex c) Leading the sample with the first and the at least one n-th bead-antibody-analyte complex into the first retention chamber and size-selective retention of the at least one first bead-antibody-analyte complex on the first retention element (14a), and passing the (n - 1)th retention element (14a) through the at least one n-th bead-antibody-analyte complex.d) guiding the sample with the at least one n-th bead-antibody-analyte complex into the n-th retention chamber (14b, 14c) and size-selective retention of the at least one n-th bead-antibody-analyte complex on the n-th retention element (14b, 14c) e) adding a specific detection reagent to 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 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 these to optionally still free specific capture antibodies (5a, 5ab, 5c).f) Adding 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 based on the detected reaction, in particular the color and / or radiation intensity, and in particular comparing this with known analyte concentrations, wherein the determination and in particular the comparison of the analyte concentrations is carried out in the evaluation unit using an evaluation algorithm, in particular. based on artificial intelligence.

11. Method according to claim 10, wherein in step b) the sample is left to rest and / or gently moved in the reaction chamber (3) for a predetermined time, in particular for 1-15 minutes.

12. 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 standing time, in particular for 0.1 - 1 min, after each pumping cycle.

13. The method according to any one of claims 10-12, wherein in steps b) and / or c) and d) at least a partial temperature control of the reaction chamber (3) and / or the retention chambers takes place, in particular at 21-40°C.

14. The 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 thyrotropin (TSH) and / or human chorionic gonadotropin (hCG) and / or anti-Müllerian hormone (AMH) and / or testosterone and / or estrogen and / or progesterone.

15. The method according to any 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 information, in particular a phase and / or a day in the female cycle and / or a fertile window of a test subject is calculated and output, in particular on a display unit and / or in an associated app.

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