Microfluidic device for handling a sample fluid and producing a plurality of aliquots from the sample fluid, method and system

Abstract

The present invention pertains to a microfluidic device (1,31) for handling at least one sample fluid (SF) and producing a plurality of aliquots (LVA,SVA) from the sample fluid (SF), the microfluidic device (1,31) comprising at least one microfluidic well (3) for receiving the at least one sample fluid (SF), wherein the microfluidic well (3) comprises at least one well inlet opening (6) for introducing the sample fluid (SF) into the microfluidic well (3), a plurality of microchambers (10,11) each for receiving an aliquot (LVA,SVA) of the plurality of aliquots (LVA,SVA) of the sample fluid (SF), and at least one well outlet opening (12) for gas displaced by the sample fluid (SF) and / or excess sample fluid (SF) to exit the microfluidic well (3), wherein the microchambers (10,11) of the microfluidic well (3) are fluidly connected to each other and to the well inlet opening (6) and the well outlet opening (12) of the microfluidic well (3) via at least one microchannel (8,9) of the microfluidic well (3), wherein the microchambers (10,11) of the at least one microfluidic well (3) comprise a plurality of large-volume microchambers (10) and a plurality of small-volume microchambers (11), and wherein the volume of each of the large-volume microchambers (10) is at least 1.5 times and at most 100000 times the volume of each of the small-volume microchambers (11).

Description

[0001]

[0002] 066093 K t8 AV2025-563 17 December 2025

[0003] QIAGEN GmbH

[0004] QIAGEN Stral e 1

[0005] 40724 Hilden

[0006] GERMANY

[0007] “Mircofluidic device for handling a sample fluid and producing a plurality of aliquots from the sample fluid, method and system"

[0008] FIELD OF THE INVENTION

[0009] The present invention relates to a microfluidic device for handling at least one sample fluid and producing a plurality of aliquots from the sample fluid, a method, preferably digital PCR method, for determining an amount and / or a concentration of a target molecule in a sample fluid, and a system comprising the microfluidic device and a handling apparatus configured to handle the microfluidic device.

[0010] BACKGROUND OF THE INVENTION

[0011] Microfluidic devices for handling sample fluids and producing a plurality of aliquots from each of the sample fluids are well known in various designs. Regardless of the respective design, such microfluidic devices typically comprise a plurality of microfluidic wells for receiving the sample fluids. In order to introduce the sample fluids into the microfluidic wells, each of the microfluidic wells usually comprises a well inlet opening. Typically, the well inlet opening is fluidly connected via at least one microchannel to a large number of microchambers of the respective microfluidic well, which are provided for receiving the aliquots of the respective sample fluid and are fluidly connected to each other via the at least one microchannel. In addition, the microchambers of each of the microfluidic wells are normally fluidly connected viathe at least one microchannel to a well outlet opening of the respective microfluidic well in order to allow gas displaced by the sample fluid and excess sample fluid to exit the microfluidic well.

[0012] Such microfluidic devices are often used in methods for determining an amount and / or a concentration of one or more target molecules such as nucleic acid molecules in a sample fluid, for example in digital polymerase chain reaction (also referred to as “digital PCR” and “dPCR”) methods. In such methods, typically a plurality of aliquots is produced from the sample fluid. Usually, the aliquots are then thermally treated in order to amplify molecules of the target molecule that may be present in the aliquots. Thereafter, typically, target molecule presence information is acquired, which indicates, for each of the thermally treated aliquots, whether or not one or more molecules of the target molecule are present in the respective aliquot. Based on the acquired target molecule presence information, the amount and / or concentration of the target molecule in the sample fluid is then determined, typically using a Poisson distribution.

[0013] Such methods for determining the amount and / or concentration of a target molecule in a sample fluid are very precise, in particular also if the relation of the target molecule to the background in the sample fluid is small. However, a disadvantage of such methods is their dynamic range, thus the ratio between the largest and smallest amount and / or concentration of target molecules that can be determined. In this respect, for example, real-time polymerase chain reaction (also referred to as “real-time PCR” and “qPCR”) methods have an advantage. In order to increase the dynamic range of dPCR methods it has already been proposed to use aliquots of different volumes as the basis for determining the amount and / or concentration of the target molecule. However, the known methods are either relatively complex and / or do not provide a satisfactory large dynamic range with precise and reliable results over the entire dynamic range.

[0014] It is therefore the object of the present invention to provide a microfluidic device, a method, and a system, preferably each of the type mentioned at the beginning and described in more detail above, which enable a large dynamic range with precise and reliable results over the entire dynamic range and little effort.SUMMARY OF THE INVENTION

[0015] According to a first aspect, the invention provides a microfluidic device for handling at least one sample fluid and producing a plurality of aliquots from the sample fluid, the microfluidic device comprising

[0016] at least one microfluidic well for receiving the at least one sample fluid,

[0017] wherein the microfluidic well comprises at least one well inlet opening for introducing the sample fluid into the microfluidic well, a plurality of microchambers each for receiving an aliquot of the plurality of aliquots of the sample fluid, and at least one well outlet opening for gas displaced by the sample fluid and / or excess sample fluid to exit the microfluidic well, wherein the microchambers of the microfluidic well are fluidly connected to each other and to the well inlet opening and the well outlet opening of the microfluidic well via at least one microchannel of the microfluidic well,

[0018] wherein the microchambers of the at least one microfluidic well comprise a plurality of large-volume microchambers and a plurality of small-volume microchambers, and wherein the volume of each of the large-volume microchambers is at least 1.5 times and at most 100000 times the volume of each of the small-volume microchambers.

[0019] By providing the plurality of large-volume microchambers and the plurality of small-volume microchambers, wherein the volume of each of the large-volume microchambers is at least 1.5 times and at most 100000 times the volume of each of the small-volume microchambers, it is possible to provide precise and reliable results over a large dynamic range with little effort. While the large-volume microchambers and the corresponding large-volume aliquots of the sample fluid ensure in particular a high sensitivity and thus that low concentrations or amounts of a target molecule in the sample fluid can be precisely and reliably determined. The smallvolume microchambers and the corresponding small-volume aliquots of the sample fluid ensure in particular that high concentrations or amounts of the target molecule in the sample fluid can be precisely and reliably determined. This is because larger aliquots enable higher sensitivity than smaller aliquots. And smaller aliquots enable the determination of higher concentrations or amounts of the target molecule before the target molecule is present in all aliquots and thus determining the concentration or amount of the target molecule in the sample fluid is no longer possible. At the same time, it is possible to ensure by means of the provided minimum and maximum relation between the respective volume of the large-volume microchambers and the respective volume of the small-volume microchambers that there isno area between the lowest and highest detectable amount or concentration where the results are unsatisfactorily unprecise and thus not reliable.

[0020] In addition to the large-volume microchambers and the small-volume microchambers, the microfluidic device can comprise further microchambers. For example, the microfluidic device can comprise a plurality of microchambers each having a larger volume than each of the large-volume microchambers, a plurality of microchambers each having a smaller volume than each of the small-volume microchambers, and / or a plurality of medium-volume microchambers each having a volume smaller than the volume of each of the large-volume microchambers and greater than the volume of each of the small-volume microchambers. In this way, the dynamic range can be further increased with precise and reliable results over the entire dynamic range. However, it can be sufficient and preferred for the sake of simplicity if the microfluidic device comprises at most 10, preferably at most 6, in particular at most 4, particularly preferably at most 3, types of microchambers, which differ with respect to the volume of the microchambers. For the same reason, it can be particularly preferred in some embodiments if the microfluidic device comprises at most 2 types of microchambers, which differ with respect to the volume of the microchambers.

[0021] A microchamber is preferably understood to mean a chamber with at least one dimension, preferably at least two perpendicular dimensions, of less than 1 mm and, preferably, at least 1 pm, for example a width, preferably diameter, and / or depth of less than 1 mm and, preferably, at least 1 pm. Accordingly, a microchannel is preferably understood to mean a channel with at least one dimension, preferably at least two perpendicular dimensions, of less than 1 mm and, preferably, at least 1 pm, for example a width and / or a depth of less than 1 mm and, preferably, at least 1 pm.

[0022] The large-volume microchambers and the small-volume microchambers do not necessarily have to have a specific volume to be large-volume microchambers or small-volume microchambers. Rather, the terms “large-volume microchambers” and “small-volume microchambers” shall express that the volume of each of the large-volume microchambers is greater than the volume of each of the small-volume microchambers.

[0023] In general, it can be sufficient if the microfluidic device comprises only one microfluidic well. However, especially in order to handle two or more different sample fluids at least partially atthe same time, which can be time-saving, it can be preferred if the microfluidic device comprises two or more microfluidic wells. Then, it is expedient if each of the microfluidic wells comprises at least one well inlet opening for introducing the respective sample fluid into the respective microfluidic well, a plurality of microchambers for receiving an aliquot of a plurality of aliquots of the respective sample fluid, and at least one well outlet opening for gas displaced by the respective sample fluid and / or excess sample fluid to exit the respective microfluidic well, wherein the microchambers of the respective microfluidic well are fluidly connected to each other and to the well inlet opening and the well outlet opening of the respective microfluidic well via at least one microchannel of the respective microfluidic well. It can be particularly expedient if the microfluidic wells are designed at least substantially identical.

[0024] Expediently, the microfluidic well, preferably each of the microfluidic wells, comprises an inlet chamber for receiving the sample fluid introduced via the well inlet opening into the microfluidic well. Then, it can be further preferred if the inlet chamber of the at least one microfluidic well comprises the well inlet opening of the microfluidic well and / or is fluidly connected via the at least one microchannel of the microfluidic well to the microchambers of the microfluidic well. For the same reason, alternatively or additionally, the microfluidic well, preferably each of the microfluidic wells, can comprise an outlet chamber for receiving excess sample fluid. Then, it can be further preferred if the outlet chamber of the at least one microfluidic well comprises the well outlet opening of the microfluidic well and / or is fluidly connected via the at least one microchannel of the microfluidic well to the microchambers of the microfluidic well.

[0025] With respect to a large dynamic range, it can be preferred if the microfluidic well, preferably each of the microfluidic wells, comprises at least 500, preferably at least 1000, in particular at least 2000, of the microchambers. A particularly large dynamic range can be achieved if the microfluidic well, preferably each of the microfluidic wells, comprises at least 5000, preferably at least 7000, in particular at least 8000, particularly preferably at least 10000, of the microchambers. In this way, in particular, a high sensitivity can be ensured. This is because with the number of the microchambers, the usable volume of the sample fluid increases and hence the lower limit of the concentration or amount of a target molecule that can be determined decreases. In contrast to this, the upper limit of the concentration or amount of a target molecule that can be determined does not increase, at least not substantially, as the number of the microchambers increases. The upper limit depends mainly on the respective volume of the small-volume microchambers. In some cases, it can also be preferred if themicrofluidic well, preferably each of the microfluidic wells, comprises at least 12500, preferably at least 15000, in particular at least 16000, of the microchambers. For example, this can be useful with respect to a large dynamic range with accurate and reliable results over the entire dynamic range if the microchambers do not comprise medium-volume microchambers with a volume between the large-volume microchambers and the small-volume microchambers. It can also be provided that the microfluidic well, preferably each of the microfluidic wells comprises at least 25000, preferably at least 40000, of the microchambers. This can be preferred, for example, if the microfluidic device has relatively few microfluidic wells, for example at most 30, and thus relatively much space is available per microfluidic well. Especially if the microfluidic device has particularly few microfluidic wells, for example at most 15, preferably at most 10, it can be even more preferred if the microfluidic well, preferably each of the microfluidic wells, comprises at least 100000, preferably at least 150000, in particular at least 180000, of the microchambers.

[0026] Irrespective of a lower limit of the number of microchambers, the microfluidic well, preferably each of the microfluidic wells, can comprise at most 500000, preferably at most 300000, in particular at most 250000, of the microchambers. This may be useful for reasons of space. It can be particularly space-saving if the microfluidic well, preferably each of the microfluidic wells, comprises at most 100000, preferably at most 75000, in particular at most 60000, of the microchambers. It can also be provided that the microfluidic well, preferably each of the microfluidic wells, comprises at most 30000, preferably at most 25000, in particular at most 20000, particularly preferably at most 15000, of the microchambers. For example, this can be sufficient with respect to a large dynamic range with accurate and reliable results over the entire dynamic range if the microchambers comprise medium-volume microchambers with a volume between the large-volume microchambers and the small-volume microchambers. Alternatively or additionally, such a maximum number of microchambers per microfluidic well can also be preferred if the microfluidic device comprises relatively many microchambers, for example at least 40, preferably at least 80. In some embodiments with medium-volume microchambers, it can even be sufficient with respect to a large dynamic range with precise and reliable results of the entire dynamic range if the microfluidic well, preferably each of the microfluidic wells, comprises at most 12000, preferably at most 10000, of the microchambers.

[0027] In a first particularly preferred embodiment of the microfluidic device, the volume of each of the large-volume microchambers is at least 100 times, preferably at least 150 times, in particularat least 180 times, the volume of each of the small-volume microchambers. In this way, the dynamic range can be further increased. Against this background, it can be even more preferred if the volume of each of the large-volume microchambers is at least 250 times, preferably at least 350 times, in particular at least 400 times, the volume of each of the smallvolume microchambers. A particularly large dynamic range can be achieved if the volume of each of the large-volume microchambers is at least 500 times, preferably at least 750 times, further preferably at least 850 times, in particular at least 900 times, the volume of each of the small-volume microchambers. Irrespective of such a lower limit, the volume of each of the large-volume microchambers can be at most 100000 times the volume of each of the smallvolume microchambers, preferably at most 50000 times the volume of each of the smallvolume microchambers, further preferably, the volume of each of the large-volume microchambers can be at most 25000 times the volume of each of the small-volume microchambers, further preferably, the volume of each of the large-volume microchambers can be at most 10000 times, preferably at most 5000 times, further preferably at most 2000 times, further preferably at most 1500 times, in particular at most 1200 times, particularly preferably at most 1000 times, the volume of each of the small-volume microchambers. In this way, it is possible to provide particularly accurate and reliable results over the entire dynamic range with little effort. Then, it can already be sufficient to provide only one type of medium-volume microchambers having at least substantially the same volume, which is smaller than the volume of each of the large-volume microchambers and greater than the volume of each of the small-volume microchambers, or even no medium-volume microchamber with a volume smaller than the volume of each of the large-volume microchambers and greater than the volume of each of the large-volume microchambers in order to achieve satisfactory results over the entire dynamic range. Regardless of this, it can also be preferred in some embodiments if the volume of each of the large-volume microchambers is at most 800 times, preferably at most 600 times, in particular at most 500 times, particularly preferably at most 450 times, the volume of each of the small-volume microchambers. In this way, a good comprise between a large dynamic range with satisfactory results over the entire dynamic range and low space requirements can be achieved, which can be particularly preferred if the microfluidic device comprises many, for example at least 80, microfluidic wells.

[0028] In a preferred embodiment, the volume of each of the large-volume microchambers is at least 0.5 nL, preferably at least 1 nL, further preferably at least 1.5 nL, in particular at least 2 nL, particularly preferably at least 2.3 nL. In this way, it can be ensured that also quite lowconcentrations and amounts of target molecules can be determined precisely and reliably by means of the microfluidic device. Against this background, it can be even more preferred if the volume of each of the large-volume microchambers is at least 5 nL, preferably at least 7 nL. Particularly low concentrations and amounts of target molecules can be determined precisely and reliably if the volume of each of the large-volume microchambers is at least 8 nL, preferably at least 8.5 nL, in particular at least 8.8 nL.

[0029] Alternatively or additionally, the volume of each of the large-volume microchambers can be at most 50 nL, preferably at most 20 nL, in particular at most 15 nL, particularly preferably at most 12 nL. This is space-saving and, at least in most cases in practice, also sufficient for a satisfactorily accurate and reliable determination of the concentration and / or amount of target molecule. Against the same background, it is even more preferred if the volume of each of the large-volume microchambers is at most 10 nL, preferably at most 9.5 nL, in particular at most 9.3 nL. In some embodiments, it can also be advantageous if the volume of each of the large-volume microchambers is at most 7 nL, preferably at most 5 nL, in particular at most 3 nL. This can be useful, for example, if the microfluidic device comprises many microfluidic wells, for example at least 80, and the space available per microfluidic well is therefore quite limited.

[0030] Irrespective of the volumes of the large-volume microchambers, the volume of each of the small-volume microchambers can be at most 0.8 nL, preferably at most 0.5 nL, in particular at most 0.4 nL. In this way, it can be ensured that quite high concentrations and amounts of target molecules can be determined precisely and reliably by means of the microfluidic device. Against this background, it is even more preferred if the volume of each of the small-volume microchambers is at most 0.1 nL, preferably at most 0.05 nL, in particular at most 0.02 nL. Particularly high concentrations and amounts of target molecules can be determined by means of the microfluidic device if the volume of each of the small-volume microchambers is at most 0.015 nL, preferably at most 0.012 nL, in particular at most 0.01 nL. In some embodiments, it can even be provided that the volume of each of the small-volume microchambers is at most 0.008 nL. This can be preferred, for example, if the microfluidic device comprises many microfluidic wells, for example at least 80, and the available space per microfluidic well is therefore very limited.

[0031] Alternatively or additionally, it can be preferred from a manufacturing point of view if the volume of each of the small-volume microchambers is at least 0.001 nL. Smaller microchambers canbe difficult to manufacture. For the same reason, it can be further preferred if the volume of each of the small-volume microchambers is at least 0.003 nL, preferably at least 0.005 nL. From a manufacturing point of view, it can be particularly preferred if the volume of each of the small-volume microchambers is at least 0.007 nL, preferably at least 0.008 nL.

[0032] Irrespective of their volume, it can be expedient if each of the large-volume microchambers has a depth of at least 100 pm, preferably at least 200 pm, in particular at least 250 pm. In some embodiments, it can be particular expedient if each of the large-volume microchambers has a depth of at least 300 pm, preferably at least 400 pm. Alternatively or additionally to such a lower limit, it can also be expedient if each of the large-volume microchambers has a depth of at most 800 pm, preferably at most 600 pm, in particular at most 500 pm. In some embodiments, it can be particular expedient if each of the large-volume microchambers has a depth of at most 400 pm, preferably at most 350 pm. Irrespective of the depth, each of the large-volume microchambers can expediently have a width, preferably a diameter, of at least 50 pm, preferably at least 100 pm, for example at least 120 pm or at least 140 pm, and / or at most 400 pm, preferably at most 300 pm, in particular at most 200 pm, particularly preferably at most 180 pm, for example at most 150 pm or at most 130 pm.

[0033] Alternatively or additionally, it can also be expedient if each of the small-volume microchambers has a depth of at least 5 pm, preferably at least 10 pm, in particular at least 15 pm. In some embodiments, it can be particularly expedient if each of the small-volume microchambers has a depth of at least 20 pm, preferably at least 25 pm. Alternatively or additionally to such a lower limit, it can also be expedient if each of the small-volume microchambers has a depth of at most 100 pm, preferably at most 50 pm, in particular at most 35 pm. In some embodiments, it can be particularly expedient if each of the small-volume microchambers has a depth of at most 30 pm, preferably at most 25 pm. Irrespective of the depth, each of the small-volume microchambers can expediently have a width, preferably a diameter, of at least 5 pm, preferably at least 10 pm, in particular at least 15 pm, and / or of at most 100 pm, preferably at most 50 pm, further preferably at most 30 pm, in particular at most 25 pm.

[0034] In a preferred embodiment, the microchambers of the at least one microfluidic well comprise a plurality of medium-volume microchambers in addition to the large-volume microchambers and the small-volume microchambers. Then, the volume of each of the medium-volumemicrochambers can be smaller than the volume of each of the large-volume microchambers and greater than the volume of each of the small-volume microchambers. This can contribute to accurate and reliable results over the entire dynamic range and can be particularly useful if there is a large size difference between the large-volume microchambers and the small-volume microchambers, for example if the volume of each of the large-volume microchambers is at least 500 times, preferably at least 750 times, in particular at least 850 times, in particular at least 900 times the volume of each of the small-volume microchambers.

[0035] Irrespective of the size ratio of the large- and small-volume microchambers, it can be expedient if the volume of each of the medium-volume microchambers is at least 0.01 nL, at least 0.1 nL, preferably at least 0.3 nL, in particular at least 0.4 nL. In some embodiments, it can be further preferred for the same reason if the volume of each of the medium-volume microchambers is at least 1 nL, preferably at least 1.5 nL, in particular at least 1.8 nL. In some embodiments, it can be particularly expedient if the volume of each of the medium-volume microchambers is at least 2.3 nL, preferably at least 2.5 nL. Irrespective of such a lower limit, it can be preferred with respect to the required sample volume if the volume of each of the medium-volume microchambers is at most 10 nL, preferably at most 5 nL, in particular at most 3.5 nL. In some embodiments, it can be further preferred for the same reason if the volume of each of the medium-volume microchambers is at most 2.5 nL, preferably at most 2.2 nL. In some embodiments, it can also be sufficient if the volume of each of the medium-volume microchambers is at most 1.5 nL, preferably at most 1 nL, in particular at most 0.8 nL. This can be particularly preferred with respect to the required sample volume.

[0036] In a preferred embodiment the volume of each of the large-volume microchambers is at least 1.5 times the volume of each of the medium-volume microchambers. This can contribute to accurate and reliable results over the entire dynamic range. For the same reason it can be even more preferred if the volume of each of the large-volume microchambers is at least 2.5, preferably at least 3 times, the volume of each of the medium-volume microchambers. In some embodiments, it can be particularly preferred for the said reason if the volume of each of the large volume-volume microchambers is at least 5 times, preferably at least 10 times, in particular at least 12 times, the volume of each of the medium-volume microchambers. Alternatively or additionally to such a lower limit, it can also be useful with respect to precise and reliable results over the entire dynamic range if the volume of each of the large-volume microchambers is at most 50 times, preferably at most 30 times, in particular at most 20 times,the volume of each of the medium-volume microchambers. In some embodiments, it can be particularly preferred for the same reason if the volume of each of the large-volume microchambers is at most 15 times, preferably at most 10 times, in particular at most 7 times, the volume of each of the medium-volume microchambers.

[0037] Regardless of the size ratio between the large-volume microchambers and the mediumvolume microchambers, the volume of each of the medium-volume microchambers can be at least 10 times, preferably at least 30 times, in particular at least 50 times, the volume of each of the small-volume microchambers. This can also contribute to precise and reliable results over the entire dynamic range. In some embodiments, it can be even more preferred for the same reason if the volume of each of the medium-volume microchambers is at least 100 times, preferably at least 150 times, in particular at least 180 times, the volume of each of the smallvolume microchambers. In some embodiments, particularly precise and reliable results can be achieved over the entire dynamic range if the volume of each of the medium-volume microchambers is at least 200 times, preferably at least 250 times, in particular at least 280 times, the volume of each of the small-volume microchambers. Alternatively or additionally to such a lower limit, it can also be useful with respect to accurate and reliable results over the whole dynamic range if the volume of each of the medium-volume microchambers is at most 500 times, preferably at most 400 times, in particular at most 350 times, particularly preferably at most 320 times, the volume of each of the small-volume microchambers. In some embodiments, it can be further preferred for the same reason if the volume of each of the medium-volume microchambers is at most 250 times, preferably at most 230 times, the volume of each of the small-volume microchambers. In some embodiments, particularly precise and reliable results can be achieved over the whole dynamic range if the volume of each of the medium-volume microchambers is at most 150 times, preferably at most 100 times, in particular at most 80 times, the volume of each of the small-volume microchambers.

[0038] In a preferred embodiment, the number of the small-volume microchambers of the microfluidic device is greater than the number of the large-volume microchambers of the microfluidic device. This can also have a positive effect on the dynamic range. A particularly large dynamic range can be achieved if the number of the small-volume microchambers of the microfluidic device is at least 1.5 times, preferably at least 1.8 times, the number of the large-volume microchambers of the microfluidic device. In some embodiments, for example if the microfluidicdevice does not comprise medium-volume microchambers having a volume smaller than the volume of each of the large-volume microchambers and greater than the volume of each of the small-volume microchambers, it can be further preferred if the number of the small-volume microchambers of the microfluidic device is at least 2 times, preferably at least 4 times, in particular at least 5 times, the number of the large-volume microchambers of the microfluidic device. In some embodiments, it can be even more preferred if the number of the small-volume microchambers of the microfluidic device is at least 10 times, preferably at least 15 times, the number of the large-volume microchambers of the microfluidic device. This can be useful, for example, if the microfluidic device comprises relatively few microfluidic wells, for example at most 30. There are also embodiments where it can be particularly advantageous if the number of the small-volume microchambers of the microfluidic device is at least 50 times, preferably at least 70 times, the number of the large-volume microchambers of the microfluidic device, for example if the microfluidic device comprises particularly few microfluidic wells, e.g. at most 15.

[0039] Alternatively or additionally, it can be expedient if the number of the small-volume microchambers of the microfluidic device is at most 200 times, preferably at most 100 times, the number of the large-volume microchambers of the microfluidic device. In some embodiments, it can be particularly expedient if the number of the small-volume microchambers of the microfluidic device is at most 50 times, preferably at most 30 times, in particular at most 25 times, the number of the large-volume microchambers of the microfluidic device. For example, if the microfluidic device comprises relatively many microfluidic wells, e.g. at least 40, it can also be preferred if the number of the small-volume microchambers of the microfluidic device is at most 15 times, preferably at most 10 times, in particular at most 8 times, the number of the large-volume microchambers of the microfluidic device. There are also embodiments where it can be particularly advantageous if the number of the small-volume microchambers of the microfluidic device is at most 6 times the number of the large-volume microchambers of the microfluidic device, for example if the microfluidic device comprises particularly many microfluidic wells, e.g. at least 80, and / or the microfluidic device comprises medium-volume microchambers.

[0040] Regardless of the ratio of the number of small-volume microchambers to the number of large-microchambers, it can be expedient if the number of the small-volume microchambers of the microfluidic device is greater than the number of the medium-volume microchambers of themicrofluidic device. For the same reason, it can be particularly preferred if the number of the small-volume microchambers of the microfluidic device is at least 2 times, preferably at least 3 times, in particular at least 4 times, the number of the medium-volume microchambers. Alternatively or additionally, it can also be particularly expedient if the number of the smallvolume microchambers of the microfluidic device is at most 30 times, preferably at most 15 times, in particular at most 10 times, the number of the medium-volume microchambers of the microfluidic device.

[0041] Irrespective of the ratio of the number of small-volume microchambers to the number of medium-volume microchambers, it can be provided that the number of the large-volume microchambers of the microfluidic device is greater than the number of the medium-volume microchambers of the microfluidic device. This can also be expedient. For the same reason it can then be further preferred if the number of the large-volume microchambers of the microfluidic device is at least 1.1 times, preferably at least 1.3 times, in particular at least 1.4 times, the number of the medium-volume microchambers of the microfluidic device. Alternatively or additionally, it can also be particularly expedient if the number of the large-volume microchambers of the microfluidic device is at most 10 times, preferably at most 5 times, in particular at most 3 times, the number of the medium-volume microchambers of the microfluidic device.

[0042] In a preferred embodiment, the large-volume microchambers have at least substantially the same volume. This can be simple and reduce the effort for manufacturing the microfluidic device. For the same reason, alternatively or additionally, the small-volume microchambers can have at least substantially the same volume. Alternatively or additionally, it can also be simple and reduce the manufacturing effort if the medium-volume microchambers have at least substantially the same volume.

[0043] Regardless of the volumes of the large-volume, small-volume and medium-volume microchambers, the microfluidic device can comprise a plurality of microfluidic wells. In this way, a plurality of different sample fluids can be handled by means of the microfluidic device at the same time, which can be time-saving. Alternatively or additionally, a sample fluid can also be split between two or more microfluidic wells, which can also be time-saving. Against this background, it can be particularly preferred if the microfluidic device comprises at least 5, preferably at least 20, further preferably at least 40, in particular at least 80, microfluidic wells.Alternatively or additionally, the microfluidic device can comprise at most 384, preferably at most 120, further preferably at most 60, in particular at most 30, particularly preferably at most 15, microfluidic wells. This can be advantageous with respect to a compact design and easy handling of the microfluidic device. A number of 8, 24, 48, or 96 microfluidic wells can be particularly preferred. Irrespective of their number, it can be expedient if each of the microfluidic wells comprises at least one well inlet opening for introducing the respective sample fluid into the respective microfluidic well, a plurality of microchambers each for receiving an aliquot of a plurality of aliquots of the respective sample fluid, and at least one well outlet opening for gas displaced by the respective sample fluid and / or excess sample fluid to exit the respective microfluidic well, wherein the microchambers of the respective microfluidic well are fluidly connected to each other and to the well inlet opening and the well outlet opening of the respective microfluidic well via at least one microchannel of the respective microfluidic well. Particularly preferably, the microfluidic wells are at least substantially of the same design. This is in particular useful if the microfluidic wells are used for different sample fluids.

[0044] In principle, it is conceivable that the large-volume microchambers on the one hand and the small-volume microchambers on the other hand are provided only in different microfluidic wells. However, in a preferred embodiment, the microfluidic well, preferably each of the microfluidic wells, comprises both a plurality of the large-volume microchambers and a plurality of the small-volume microchambers. This can be preferred with respect to easy handling of the microfluidic device. Then, the at least one sample fluid does not have to be split between different microfluidic wells. Against this background, it can be further preferred if the microfluidic well, preferably each of the microfluidic wells, comprises also a plurality of the medium-volume microchambers in addition to the large-volume microchambers and the small-volume microchambers. In particular if the microfluidic device comprises only one microfluidic well, the microfluidic well can, for the sake of simplicity, comprise all the large-volume microchambers and all the small-volume microchambers and, optionally, all the medium-volume microchambers of the microfluidic device. However, if the microfluidic device comprises more than one microfluidic well, it can be preferred for the said reason if each of the microfluidic wells comprises a plurality of the large-volume microchambers and a plurality of the smallvolume microchambers and, optionally, a plurality of the medium-volume microchambers.

[0045] With respect to reliable results, it can be preferred if the microfluidic well, preferably each of the microfluidic wells, comprises at least 500 of the large-volume microchambers. Particularreliable results can be ensured if the microfluidic well, preferably each of the microfluidic wells, comprises at least 800, preferably at least 1000, of the large-volume microchambers. Irrespective of the number of large-volume microchambers, it can be preferred for the same reason if the microfluidic well, preferably each of the microfluidic wells, comprises at least 500, preferably at least 800, in particular at least 1000, of the small-volume microchambers. Alternatively or additionally, it can also contribute to reliable results if the microfluidic well, preferably each of the microfluidic wells, comprises at least 500, preferably at least 800, in particular at least 1000, of the medium-volume microchambers.

[0046] In a preferred embodiment the microfluidic well, preferably each of the microfluidic wells, comprises at least 1500, preferably at least 1800, in particular at least 1900, of the large-volume microchambers. This can be advantageous with respect to the lowest concentration or amount of a target molecule that can be determined using one microfluidic well. In this respect, it can be particularly preferred if the number of the large-volume microchambers of the microfluidic well, preferably of each of the microfluidic wells, is at least 2300, preferably at least 2400. In some embodiments, for example if the microfluidic well does not comprise mediumvolume microchambers having a volume smaller than the volume of each of the large-volume microchambers and bigger than the volume of each of the small-volume microchambers, it can be even more preferred with respect to the lowest determinable concentration or amount of target molecule if the microfluidic well, preferably each of the microfluidic wells, comprises at least 2800, preferably at least 2900, of the large-volume microchambers. Irrespective of a lower limit, it can be provided that the microfluidic well, preferably each of the microfluidic wells, comprises at most 10000, preferably at most 5000, in particular at most 3500, particularly preferably at most 3200, of the large-volume microchambers. The invention has discovered that a respective number of large-volume microchambers can be sufficient and that further increasing the number of the large-volume microchambers leads at most to an insignificant increase in the lowest detectable amount or concentration of a target molecule in relation to the additional volume of sample fluid required to fill the additional large-volume microchambers. If not enough sample fluid is available to fill all the microchambers, this would even lower the sensitivity. Against this background, it can be particularly preferred if the number of the large-volume microchambers of the microfluidic well, preferably of each of the microfluidic wells, is at most 2800, preferably at most 2600. In some embodiments, it can be even more preferred if the microfluidic well, preferably each of the microfluidic wells, comprises at most 2300, preferably at most 2100, of the large-volume microchambers.With respect to the highest amount or concentration of a target molecule that can be determined by means of one microfluidic well, it can be advantageous if the microfluidic well, preferably each of the microfluidic wells, comprises at least 2000, preferably at least 4000, in particular at least 4500, of the small-volume microchambers. In some embodiments, it can be particularly preferred for the same reason if the microfluidic well, preferably each of the microfluidic wells, comprises at least 8000, preferably at least 9000, of the small-volume microchambers. For example, if the microfluidic well does not comprise medium-volume microchambers having a volume smaller than the volume of each of the large-volume microchambers and bigger than the volume of each of the small-volume microchambers, it can be further preferred if the number of the small-volume microchambers of the microfluidic well, preferably of each of the microfluidic wells, is at least 13000, preferably at least 14000. In some embodiments, for example if the microfluidic device comprises relatively few microfluidic wells, e.g. at most 30, it can also be preferred if the microfluidic well, preferably each of the microfluidic wells, comprises at least 25000, preferably at least 40000, in particular at least 45000, of the small-volume microchambers. Especially if the microfluidic device comprises particularly few microfluidic wells, for example at most 15, it can be even more preferred if the microfluidic well, preferably each of the microfluidic wells, comprises at least 150000, preferably at least 180000, in particular at least 200000, of the small-volume microchambers.

[0047] Irrespective of a lower limit, it can be provided that the microfluidic well, preferably each of the microfluidic wells, comprises at most 500000, preferably at most 300000, further preferably at most 250000, in particular at most 220000, of the small-volume microchambers. This is spacesaving and allows determining of sufficiently high amounts and concentrations of target molecules at least in most cases in practice. Against this background, it can be particularly preferred in some embodiments if the number of the small-volume microchambers of the microfluidic well, preferably of each of the microfluidic wells, is at most 100000, preferably at most 60000, in particular at most 55000. For example, if the microfluidic device comprises relatively many microfluidic wells, e.g. at least 40, it can be further preferred for the same reason if the microfluidic well, preferably each of the microfluidic wells, comprises at most 30000, preferably at most 20000, in particular at most 17000, particularly preferably at most 16000, of the small-volume microchambers. In some embodiments, it can also be provided that the microfluidic well, preferably each of the microfluidic wells, comprises at most 12000, preferably at most 11000, of the small-volume microchambers. This can be preferred, forexample, if the microfluidic well comprises medium-volume microchambers having a volume smaller than the volume of each of the large-volume microchambers and greater than the volume of each of the small-volume microchambers and / or the microfluidic device comprises particularly many, e.g. at least 80, microfluidic wells. Especially if the microfluidic well comprises medium-volume microchambers, it can be particularly preferred for space reasons if the microfluidic well, preferably each of the microfluidic well comprises at most 7000, preferably at most 6000, in particular at most 5500, of the small-volume microchambers.

[0048] Regardless of the number of the small-volume microchambers per microfluidic well, it can be advantageous if the microfluidic well, preferably each of the microfluidic wells, comprises at most 5000 of the medium-volume microchambers. This can be space-saving and sufficient in order to ensure accurate and reliable results over the entire dynamic range. For the same reason, it can then be particularly preferred if the microfluidic well, preferably each of the microfluidic wells, comprises at most 3000, preferably at most 2000, in particular at most 1800, for example at most 1500, of the medium-volume microchambers.

[0049] In a preferred embodiment, the microfluidic well comprises at least one large-volume subwell comprising the large-volume microchambers of the microfluidic well and at least one smallvolume subwell comprising the small-volume microchambers of the microfluidic well. This can simplify the control of the filling of the microchambers and contribute to reliable detection of whether or not one or more of a target molecule are present in the microchambers. If the microfluidic device comprises two or more microfluidic wells, it can be preferred for the same reasons if each of the microfluidic wells comprises at least one large-volume subwell comprising the large-volume microchambers of the respective microfluidic well and at least one small-volume subwell comprising the small-volume microchambers of the respective microfluidic well. Alternatively or additionally, it can be particularly preferred for the said reasons if the microfluidic well, preferably each of the microfluidic wells, comprises also at least one medium-volume subwell comprising the medium-volume microchambers in addition to the at least one large-volume subwell and the at least one small-volume subwell. Irrespective of this, each of the subwells can expediently comprise also channel segments of the at least one microchannel which fluidly connect the microchambers of the respective subwell. Alternatively or additionally, the at least one small-volume subwell and the at least one large-volume subwell and, optionally, the at least one medium-volume subwell of the microfluidic well, preferably of each of the microfluidic wells, can expediently be arranged adjacent to each other or spacedapart from each other. Then, preferably, none of the microchambers of one of the subwells is arranged between two or more of the microchambers of one of the at least one other subwell.

[0050] The volume of the large-volume subwell does not necessarily have to be greater than the volume of the at least one small-volume subwell. Likewise, the volume of the at least one medium-volume subwell does not necessarily have to be greater than the volume of the at least one small-volume subwell or smaller than the volume of the at least one large-volume subwell. Rather, the terms “large-volume subwell”, “small-volume subwell” and “mediumvolume subwell” indicate which microchambers the respective subwell comprises, namely large-volume microchambers, small-volume microchambers, or medium-volume microchambers. However, nevertheless, it can be expedient if the volume of the large-volume subwell, preferably of each of the large-volume subwells, is greater than the volume of the small-volume subwell, preferably of each of the small-volume subwells and / or if the volume of the medium-volume subwell, preferably of each of the medium-volume subwells, is smaller than the volume of the large-volume subwell, preferably of each of the large-volume subwells, and / or greater than the volume of the small-volume subwell, preferably of each of the smallvolume subwells. Irrespective of this, it can be simple and expedient if the microfluidic well, preferably each of the microfluidic wells, comprises only one large-volume subwell and only one small-volume subwell and, optionally, only one medium-volume subwell.

[0051] With respect to a simple design of the microfluidic device and simple control of the filling of the microchambers, it can be advantageous if the at least one large-volume subwell of the microfluidic well is fluidly connected to the well outlet opening or the well inlet opening of the microfluidic well, preferably only, via the at least one small-volume subwell and / or the at least one medium-volume subwell of the microfluidic well. A fluidic connection to the well outlet opening via the at least one small-volume subwell and / or the at least one medium-volume subwell can be particularly preferred. For the same reason, alternatively or additionally, the at least one small-volume subwell of the microfluidic well can be fluidly connected to the well inlet opening or the well outlet opening of the microfluidic well, preferably only, via the at least one large-volume subwell and / or the at least one medium-volume subwell of the microfluidic well. A fluidic connection to the well inlet opening via the at least one large-volume subwell and / or the at least one medium-volume subwell can be particularly preferred. If the microfluidic device comprises two or more microfluidic wells, it can also be preferred for the said reason if the at least one large-volume subwell of each of the microfluidic wells is fluidly connected to the welloutlet opening or the well inlet opening of the respective microfluidic well via the at least one small-volume subwell and / or the at least one medium-volume subwell of the respective microfluidic well and / or the at least one small-volume subwell of each of the microfluidic wells is fluidly connected to the well inlet opening or the well outlet opening of the respective microfluidic well via the at least one large-volume subwell and / or the at least one mediumvolume subwell of the respective microfluidic well.

[0052] Irrespective of a fluidic connection to the well inlet opening or the well outlet opening via one or more of the other subwells, it can be preferred if the at least one large-volume subwell is fluidly connected to the at least one small-volume subwell via at least one channel branch for dividing a fluid flow of the sample fluid from the large-volume subwell into at least two, preferably at least three, in particular at least four, fluid flows and / or for combining at least two, preferably at least three, in particular at least four, separate fluid flows of the sample fluid from the small-volume subwell into one fluid flow. This can have a positive effect on the time required to fill the microchambers with the sample fluid. For the same reason, it can be preferred alternatively or additionally if the at least one medium-volume subwell is fluidly connected to the at least one small-volume subwell via at least one channel branch for dividing a fluid flow of the sample fluid from the medium-volume subwell into at least two, preferably at least three, in particular at least four, fluid flows and / or for combining at least two, preferably at least three, in particular at least four, separate fluid flows from the small-volume subwell into one fluid flow. Irrespective of this, it can also have a positive effect on the time required to fill the microchambers if the at least one medium-volume subwell is fluidly connected to the at least one large-volume subwell via at least one channel branch for dividing a fluid flow of the sample fluid from the large-volume subwell into at least two, preferably at least three, in particular at least four, fluid flows and / or for combining at least two, preferably at least three, in particular at least four, separate fluid flows from the medium-volume subwell into one fluid flow.

[0053] In a preferred embodiment, the microfluidic device comprises a sealing layer for fluidly separating the microchambers of the at least one microfluidic well from each other by sealing channel segments of the at least one microchannel that fluidly connect the microchambers to each other. This allows easy and reliable fluidic separation of the microchambers and thus of the aliquots contained therein. If the microfluidic device comprises two or more microfluidic wells, it can be preferred for the same reason if the sealing layer is designed for fluidly separating the microchambers of each of the microfluidic wells from each other by sealingchannel segments of the at least one microchannel of the respective microfluidic well that fluidly connect the microchambers to each other. Irrespective of this, the sealing layer can be designed for sealing the at least one microchannel at least substantially entirely. However, this is not absolutely necessary. Alternatively or additionally, it can be expedient if the sealing layer covers the at least one microfluidic well, preferably each of the microfluidic wells, at least partially, preferably at least substantially.

[0054] A particular simple and yet reliable fluidic separation of the microchambers can be achieved if the sealing layer is designed for fluidly separating the microchambers of the at least one microfluidic well by deformation, for example elastic deformation and / or plastic deformation, into the channel segments of the at least one microchannel that fluidly connect the microchambers to each other. A deformation of the sealing layer into the channel segments can be achieved, for example, by heating the sealing layer. However, fluidic separation of the microchambers can be achieved particularly easily and in particular reliably if the sealing layer is designed for deforming into the channel segments upon application of a compressive force to the sealing layer. As a result of the applied compressive force, the sealing layer can then be pressed into the channel segments. The compressive force can for example be applied to the sealing layer by means of a roller and / or a clamping plate.

[0055] It can be simple and expedient if the sealing layer comprises a film, preferably a plastic film. For the same reason, it can be even more preferred if the sealing layer is at least substantially formed by the film, in particular plastic film. Irrespective of a film, it can be expedient if the sealing layer is attached to a base body of the microfluidic device which forms the at least one microfluidic well. In particular, the sealing layer can be glued to the base body. This can be particularly simple and expedient. In particular if the sealing layer is glued to the base body, but also in general it can be preferred if the sealing layer is an adhesive sealing layer. This can simplify the manufacture of the microfluidic device. Irrespective of a film, the sealing layer can be at least partially, preferably at least substantially, transparent. This can be expedient.

[0056] In a preferred embodiment, the microfluidic device comprises a protective layer which seals the at least one well inlet opening and / or the at least one well outlet opening. This can help to protect the sample fluid in the at least one microfluidic well against contamination with foreign matter and the surroundings of the microfluidic device against contamination with the sample fluid.The protective layer can be at least partially gas permeable. Then, gas can be fed via a gas flow through the protective layer into the well inlet opening and / or gas can be sucked via a gas flow through the protective layer out of the well outlet opening, thereby generating a filling pressure in the form of an overpressure at the well inlet opening and / or an underpressure at the well outlet opening, as a result of which the sample fluid within the microfluidic well can flow into the microchambers of the microfluidic well. In this way, the filling pressure applied to the sample fluid can be controlled very precisely, thus allowing a very reliable filling of the microchambers with the sample fluid, while reliably preventing contamination of the sample fluid and the surroundings of the microfluidic device. Against this background, it can be sufficient if the protective layer is gas permeable at least in one or more sections adjacent to the at least one well inlet opening and / or the at least one well outlet opening. However, for the sake of simplicity, it can be preferred if the entire protective layer is gas permeable. Then, the same protective layer can also be used for different arrangements of the at least one well inlet opening and / or well outlet opening. In principle irrespective of whether the protective layer is partially or entirely permeable to gas, it can be preferred if the protective layer comprises a gas permeable material. Then, the gas permeability of the protective layer can be provided by the gas permeable material. This can be simple and expedient. Alternatively or additionally to a gas permeable material, the protective layer can also comprise one or more inlet valves and / or outlet valves. Then, the at least one valve can provide for the gas permeability of the protective layer. This is more effortful, but can be preferred due to a reduced gas resistance. The at least one valve can for example be a Duckbill vale.

[0057] Alternatively or additionally to a gas permeability, the protective layer can be designed at least partially such that it reseals itself after being penetrated, for example with a needle. In this way, the protective layer can seal the well inlet opening and / or the well outlet opening reliably even if it is necessary to penetrate the protective layer during use of the microfluidic device. For example, it is possible in this way to apply the protective layer before the sample fluid is introduced into the at least one microfluidic well through the protective layer, for example by means of a needle, and still ensure a reliable protection against contamination by the protective layer thereafter. Against this background, it can be sufficient if the protective layer is designed in one or more sections adjacent to the at least one well inlet opening such that it reseals itself after being penetrated. Irrespective of this, the protective layer can for example comprise at least one valve, for example a Duckbill valve, which reseals itself after being penetrated. Then,the resealability of the protective layer can be provided by the at least one valve. However, it can be particularly simple and expedient if the protective layer comprises a material, preferably an elastomer, which reseals itself after being penetrated, for example with a needle. Such elastomers are known from medicine, for example, where they are used in ampules. The elastomer can, for example, be provided in the form of a rubber coating.

[0058] Irrespective of a resealability, the protective layer can comprise a film, preferably a plastic film. This can be simple and expedient. For the same reason, it can be even more preferred if the protective layer is at least substantially formed by the film, in particular plastic film. Alternatively or additionally to a film, it can also be expedient if the protective layer is attached, preferably glued to a base body of the microfluidic device which forms the at least one microfluidic well. In principle, it is conceivable that the protective layer and the sealing layer are provided on the same side of the base body. However, it can be preferred if the sealing layer and the protective layer are arranged on opposite sides of the base body. This can simplify the design of the microfluidic device. Irrespective of this and in particular if the protective layer is glued to the base body, but also in general it can be preferred if the protective layer is an adhesive protective layer. This can simplify the manufacture of the microfluidic device.

[0059] According to a second aspect, the invention provides a method, preferably digital PCR method, for determining an amount and / or a concentration of a target molecule, preferably nucleic acid molecule, in a sample fluid, preferably using the microfluidic device according to the first aspect, the method comprising

[0060] producing a plurality of aliquots from the sample fluid, wherein the aliquots comprise a plurality of large-volume aliquots and a plurality of small-volume aliquots, wherein the volume of each of the large-volume aliquots is at least 1.5 times and at most 100000 times the volume of each of the small-volume aliquots,

[0061] acquiring target molecule presence information, wherein the target molecule presence information is indicative, for each of the aliquots, of whether or not the respective aliquot comprises one or more molecules of the target molecule, and

[0062] determining the amount and / or concentration of the target molecule in the sample fluid based on the acquired target molecule presence information.

[0063] The advantages of the method according to the second aspect are the same as those explained above in connection with the microfluidic device according to the first aspect. Inparticular, the use of large-volume aliquots and small-volume aliquots, wherein the volume of each of the large-volume aliquots is at least 1.5 times and at most 100000 times the volume of each of the small-volume aliquots, for the determination of the amount and / or concentration of a single target molecule in a single sample fluid allows for precise and reliable results over a large dynamic range with little effort.

[0064] The sample fluid can expediently comprise a, preferably biological and / or fluidic, sample. In addition to the sample, the sample fluid can expediently comprise one or more further components, for example one or more reagents.

[0065] In the method, the microfluidic device according to the first aspect can be used. In particular, the plurality of aliquots of the sample fluid can be produced by means of the microfluidic device and the target molecule presence information can be acquired while the aliquots are in the respective microchambers of the microfluidic device. Alternatively to using the microfluidic device, the method can be a droplet digital PCR method, even though this is not preferred.

[0066] In addition to the large-volume aliquots and the small-volume aliquots, further aliquots can be produced from the sample fluid, for example a plurality of aliquots each having a larger volume than each of the large-volume aliquots, a plurality of aliquots each having a smaller volume than each of the small-volume aliquots, and / or a plurality of medium-volume aliquots each having a volume smaller than the volume of each of the large-volume aliquots and greater than the volume of each of the small-volume aliquots. In this way, the dynamic range can be further increased with precise and reliable results of the entire dynamic range. However, it can be sufficient with respect to the dynamic range and preferred for the sake of simplicity if the aliquots produced from the sample fluid comprise at most 10, preferably at most 6, in particular at most 4, particularly preferably at most 3, types of aliquots, which differ with respect to the volume of the aliquots. For the same reason, it can be particularly preferred in some embodiments if the aliquots produced from the sample fluid comprise only the large-volume aliquots and the small-volume aliquots.

[0067] With respect to a large dynamic range, it can be preferred if the plurality of aliquots produced from the sample fluid comprise at least 500, at least 1000, at least 2000, at least 5000, at least 7000, at least 8000, or at least 10000 aliquots. In some embodiments, it can also be preferred if the plurality of aliquots produced from the sample fluid comprise at least 12500, at least15000, at least 16000, at least 25000, at least 40000, at least 100000, at least 150000, or at least 180000 aliquots. Alternatively or additionally to a lower limit, it can be sufficient if the plurality of aliquots produced from the sample fluid are at most 500000, at most 300000, at most 250000, at most 100000, at most 75000, or at most 60000 aliquots. In some embodiments, it can also be preferred if the plurality of aliquots produced from the sample fluid are at most 30000, at most 25000, at most 20000, at most 15000, at most 12000, or at most 10000 aliquots.

[0068] Irrespective of the total number of aliquots, it can be preferred if the plurality of aliquots comprise at least 500, at least 800, at least 1000, at least 1500, at least 1800, at least 1900, at least 2300, at least 2400, at least 2800, or at least 2900 of the large-volume aliquots. Alternatively or additionally, the plurality of aliquots can comprise at most 10000, at most 5000, at most 3500, at most 3200, at most 2800, at most 2600, at most 2300, or at most 2100 of the large-volume aliquots. Irrespective of the number of large-volume aliquots, the plurality of aliquots can comprise at least 2000, at least 4000, at least 4500, at least 8000, at least 9000, at least 13000, at least 14000, at least 25000, at least 40000, at least 45000, at least 150000, at least 180000, or at least 200000 of the small-volume aliquots. Alternatively or additionally, it can also be preferred if the plurality of aliquots comprise at most 500000, at most 300000, at most 250000, at most 220000, at most 100000, at most 60000, at most 55000, at most 30000, at most 20000, at most 17000, at most 16000, at most 12000, at most 11000, at most 7000, at most 6000, or at most 5500 of the small-volume aliquots. The advantages of these embodiments are principally the same as those described above with respect to the microfluidic device according to the first aspect in connection with the numbers of the large-volume microchambers and the small-volume microchambers.

[0069] The target molecule presence information can simply and expediently be acquired by at least one sensor, preferably image sensor, for example camera. Then, the target molecule presence information can represent one or more images of one or more of the aliquots, preferably within the respective microchambers of the microfluidic device. Preferably, the target molecule presence information can be acquired after the aliquots have been and / or while the aliquots are illuminated with excitation light, preferably with one or more wavelengths from the ultraviolet range. The target molecule presence information can then be indicative, for each of the aliquots, of whether or not the respective aliquot fluoresces, wherein it can indicate thepresence of one or more molecules of the target molecule in the respective aliquot if the aliquot fluoresces.

[0070] Expediently, the amount and / or concentration of the target molecule in the sample fluid can be determined based on further information in addition to the target molecule presence information, for example aliquot validity information, which can be indicative, for each of the aliquots, of whether or not the respective aliquot is valid, and / or aliquot volume information, which can be indicative of the volume of each of the aliquots. Irrespective of this, the amount and / or concentration of the target molecule in the sample fluid can simply and expediently be determined by a data processing unit.

[0071] The large-volume aliquots and the small-volume aliquots do not necessarily have to have a specific volume to be large-volume aliquots or small-volume aliquots. Rather, the terms “large-volume aliquots” and “small-volume aliquots” shall express that the volume of each of the large-volume aliquots is greater than the volume of each of the small-volume aliquots.

[0072] In a first particularly preferred embodiment of the method, the volume of each of the large-volume aliquots is at least 100 times, preferably at least 150 times, in particular at least 180 times, the volume of each of the small-volume aliquots. In this way, the dynamic range can be further increased. For the same reason, it can be even more preferred if the volume of each of the large-volume aliquots is at least 250 times, preferably at least 350 times, in particular at least 400 times, the volume of each of the small-volume aliquots. A particularly large dynamic range can be achieved if the volume of each of the large-volume aliquots is at least 500 times, preferably at least 750 times, further preferably at least 850 times, in particular at least 900 times, the volume of each of the small-volume microchambers. Irrespective of the lower limit, the volume of each of the large-volume aliquots can be at most 100000 times the volume of each of the small-volume aliquots, preferably at most 50000 times the volume of each of the small-volume aliquots, further preferably, the volume of each of the large-volume aliquots can be at most 25000 times the volume of each of the small-volume aliquots, further preferably, the volume of each of the large-volume aliquots can be at most 10000 times, further preferably at most 5000 times, further preferably at most 2000 times, further preferably at most 1500 times, in particular at most 1200 times, particularly preferably at most 1000 times, the volume of each of the small-volume aliquots. In this way, it is possible to provide particularly accurate and reliable results over the entire dynamic range with little effort. In some embodiments, it canalso be preferred if the volume of each of the large-volume aliquots is at most 800 times, preferably at most 600 times, in particular at most 500 times, particularly preferably at most 450 times, the volume of each of the small-volume microchambers. In this way, a good compromise between a large dynamic range with satisfactory results over the entire dynamic range and little effort can be achieved.

[0073] Irrespective of the relation of the volumes of the large-volume aliquots and the small-volume aliquots, the volume of each of the large-volume aliquots can be at least 0.5 nL, at least 1 nL, at least 1.5 nL, at least 2 nL, at least 2.3 nL, at least 5 nL, at least 7 nL, at least 8 nL, at least 8.5 nL, or at least 8.8 nL. Alternatively or additionally, the volume of each of the large-volume aliquots can be at most 50 nL, at most 20 nL, at most 15 nL, at most 12 nL, at most 10 nL, at most 9.5 nL, at most 9.3 nL, at most 7 nL, at most 5 nL, or at most 3 nL. The advantages of these embodiments are principally the same as those described above with respect to microfluidic device according to the first aspect in connection with the volumes of the large-volume microchambers.

[0074] The volume of each of the small-volume aliquots can be at most 0.8 nL, at most 0.5 nL, at most 0.4 nL, at most 0.1 nL, at most 0.05 nL, at most 0.02 nL, at most 0.015 nL, at most 0.012 nL, at most 0.01 nL, or at most 0.008 nL. Alternatively or additionally, the volume of each of the small-volume aliquots can be at least 0.001 nL, at least 0.003 nL, at least 0.005 nL, at least 0.007 nL, or at least 0.008 nL. The advantages of these embodiments are principally the same as those described above with respect to the microfluidic device according to the first aspect in connection with the volumes of the small-volume microchambers.

[0075] Irrespective of their specific volume, it can be simple and expedient if the large-volume aliquots have at least substantially the same volume. For the same reason, alternatively or additionally, the small-volume aliquots can have at least substantially the same volume.

[0076] The number of the small-volume aliquots can be greater than the number of the large-volume aliquots. Preferably, the number of the small-volume aliquots is at least 1.5 times, at least 1.8 times, at least 2 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 50 times, or at least 70 times the number of the large-volume aliquots. Alternatively or additionally, the number of the small-volume aliquots can be at most 200 times, most 100 times, at most 50 times, at most 30 times, at most 25 times, at most 15 times, at most 10 times,at most 8 times, or at most 6 times the number of the large-volume aliquots. The advantages of these embodiments are principally the same as those described above with respect to the microfluidic device according to the first aspect in connection with the relation between the numbers of the small-volume microchambers and the large-volume microchambers.

[0077] In a preferred embodiment, the aliquots produced from the sample fluid comprise a plurality of medium-volume aliquots, wherein the volume of each of the medium-volume aliquots is smaller than the volume of each of the large-volume aliquots and greater than the volume of each of the small-volume aliquots. This can contribute to accurate and reliable results over the entire dynamic range and can be particularly useful if there is a large size difference between the large-volume aliquots and the small-volume aliquots, for example if the volume of each of the large-volume aliquots is at least 500 times, preferably at least 750 times, in particular at least 850 times, the volume of each of the small-volume aliquots. Irrespective of this, it can be preferred for the sake of simplicity if the medium-volume aliquots have at least substantially the same volume.

[0078] Irrespective of the size ratio of the large- and small-volume aliquots, it can contribute to accurate and reliable results over the entire dynamic range if the volume of each of the large-volume aliquots is at least 1.5 times, preferably at least 2.5 times, in particular at least 3 times, the volume of each of the medium-volume aliquots. In some embodiments it can be particularly preferred for the same reason if the volume of each of the large-volume aliquots is at least 5 times, preferably at least 10 times, in particular at least 12 times, the volume of each of the medium-volume aliquots. Alternatively or additionally to such a lower limit, it can also be useful with respect to precise and reliable results over the entire dynamic range if the volume of each of the large-volume aliquots is at most 50 times, preferably at most 30 times, in particular at most 20 times, the volume of each of the medium-volume aliquots. In some embodiments, it can be particularly preferred for the same reason if the volume of each of the large-volume aliquots is at most 15 times, preferably at most 10 times, in particular at most 7 times, the volume of each of the medium-volume aliquots.

[0079] Regardless of the size ratio between the large-volume aliquots and the medium-volume aliquots, the volume of each of the medium-volume aliquots can be at least 10 times, preferably at least 30 times, in particular at least 50 times, the volume of each of the small-volume aliquots. This can also contribute to precise and reliable results over the entire dynamic range.In some embodiments it can be even more preferred for the same reason if the volume of each of the medium-volume aliquots is at least 100 times, preferably at least 150 times, in particular at least 180 times, the volume of each of the small-volume aliquots. In some embodiments, particularly precise and reliable results can be achieved over the entire dynamic range if the volume of each of the medium-volume aliquots is at least 200 times, preferably at least 250 times, in particular at least 280 times, the volume of each of the small-volume aliquots. Alternatively or additionally to such a lower limit, it can also be useful with respect to accurate and reliable results over the whole dynamic range if the volume of each of the medium-volume aliquots is at most 500 times, preferably at most 400 times, in particular at most 350 times, particularly preferably at most 320 times, the volume of each of the small-volume aliquots. In some embodiments, it can be further preferred for the same reason if the volume of each of the medium-volume aliquots is at most 250 times, preferably at most 230 times, the volume of each of the small-volume aliquots. In some embodiments, particularly precise and reliable results can be achieved over the whole dynamic range if the volume of each of the mediumvolume aliquots is at most 150 times, preferably at most 100 times, in particular at most 80 times, the volume of each of the small-volume aliquots.

[0080] The volume of each of the medium-volume aliquots can be, for example, at least 0.01 nL, at least 0.1 nL, at least 0.3 nL, at least 0.4 nL, at least 1 nL, at least 1.5 nL, at least 1.8 nL, at least 2.3 nL, or at least 2.5 nL. Alternatively or additionally, the volume of each of the mediumvolume aliquots can be at most 10 nL, at most 5 nL, at most 3.5 nL, at most 2.5 nL, at most 2.2 nL, at most 1.5 nL, at most 1 nL, or at most 0.8 nL. The advantages of these embodiments are principally the same as those described above with respect to microfluidic device according to the first aspect in connection with the volumes of the medium-volume microchambers.

[0081] Regardless of the volumes of the medium-volume aliquots, the number of the small-volume aliquots can be greater than the number of the medium-volume aliquots, preferably at least 2 times, in particular at least 3 times, particularly preferably at least 4 times, and / or at most 30 times, in particular at most 15 times, particularly preferably at most 10 times, the number of the medium-volume aliquots. Alternatively or additionally, the number of the large-volume aliquots can be greater than the number of the medium-volume aliquots, preferably at least 1.1 times, in particular at least 1.3 times, particularly preferably at least 1.4 times, and / or at most 10 times, in particular at most 5 times, particularly preferably at most 3 times, the number of the medium-volume aliquots. The advantages of these embodiments are principally the sameas those described above with respect to the microfluidic device according to the first aspect in connection with the ratios of the number of the medium-volume microchambers, the large-volume microchambers, and the small-volume microchambers.

[0082] Preferably, the plurality of aliquots comprise at least 500, at least 800, or at least 1000 of the medium-volume aliquots. Alternatively or additionally, the plurality of aliquots can comprise at most 5000, at most 3000, at most 2000, at most 1800, or at most 1500 of the medium-volume aliquots. The advantages of these embodiments are principally the same as those described above with respect to the microfluidic device according to the first aspect in connection with the number of the medium-volume microchambers.

[0083] In a preferred embodiment, the producing the plurality of aliquots from the sample fluid comprises applying a filling pressure to the sample fluid within the at least one microfluidic well of the microfluidic device such that the sample fluid flows into at least a plurality of the large-volume microchambers and into at least a plurality of the small-volume microchambers and, preferably, at least a plurality of the medium-volume microchambers of the at least one microfluidic well as a result of the applied filling pressure.

[0084] In this way, the microchambers can be reliably filled with the sample fluid in a short time. Against this background, it can be even more preferred if the filling pressure is applied to the sample fluid within the at least one microfluidic well such that the sample fluid flows into at least a majority of, preferably at least substantially all, the large-volume microchambers and / or into at least a majority of, preferably at least substantially all, the small-volume microchambers of the at least one microfluidic well as a result of the applied filling pressure. However, although it should not be excluded, it is not absolutely necessary that all of the microchambers of the at least one microfluidic well are filled with sample fluid as a result of the applied filling pressure. Rather it can be preferred with respect to an economic use of the sample fluid if one or more of the microchambers of the at least one microfluidic well are free from sample fluid after the application of the filling pressure. The one or more microchambers that are free from sample fluid after the application of the filling pressure can then be filled with sample fluid in a subsequent method step, preferably as a result of the fluidic separation of the microchambers of the at least one microfluidic well from each other. Irrespective of this, the filling pressure applied to the sample fluid within the at least one microfluidic well can expediently comprise an overpressure generated at an inlet side of the microfluidic well assigned to the well inletopening and / or an underpressure generated at an outlet side of the microfluidic well assigned to the well outlet opening.

[0085] With respect to a precise and reliable filling of the microchambers with sample fluid, it can be preferred if the filling pressure is applied at different, preferably at least substantially constant, pressure levels to the sample fluid within the at least one microfluidic well when filling the large-volume microchambers with the sample fluid on the one hand and when filling the small-volume microchambers with the sample fluid on the other hand. For the same reason, it can be further preferred if the filling pressure is also applied at a, preferably at least substantially constant, pressure level to the sample fluid within the at least one microfluidic well when filling the medium-volume microchambers with the sample fluid, which is different from the pressure levels when filling the large-volume microchambers and when filling the small-volume microchambers with the sample fluid. Irrespective of this, it can then be preferred for the sake of simplicity if the filling pressure is applied at the different pressure levels to the sample fluid when filling the at least one large-volume subwell with the sample fluid on the one hand and when filling the at least one small-volume subwell with the sample fluid on the other hand and, preferably, when filling the at least one medium-volume subwell. A particular precise and reliable filling of the microchambers can be achieved if the overpressure generated at the inlet side of the at least one microfluidic well and / or the underpressure generated at the outlet side of the at least one microfluidic well is greater when filling the large-volume microchambers, preferably the at least one large-volume subwell, with the sample fluid than when filling the small-volume microchambers, preferably the at least one small-volume subwell, with the sample fluid and / or when filling the medium-volume microchambers, preferably the at least one medium-volume subwell, with the sample fluid. For the same reason, it can alternatively or additionally also be useful for the said reason if the overpressure generated at the inlet side of the at least one microfluidic well and / or the underpressure generated at the outlet side of the at least one microfluidic well is greater when filling the medium-volume microchambers, preferably the at least one medium-volume subwell, with the sample fluid than when filling the small-volume microchambers, preferably the at least one small-volume subwell, with the sample fluid.

[0086] Alternatively to different pressure levels, the filling pressure can be applied at, at least substantially, the same, preferably at least substantially constant, pressure level to the sample fluid within the at least one microfluidic well while filling the large-volume microchambers andwhile filling the small-volume microchambers and, preferably, while filling the medium-volume microchambers with the sample fluid. This makes controlling the filling process particularly easy. For the same reason, it can then be further preferred if the filling pressure is applied at, at least substantially, the same, preferably at least substantially constant, pressure level to the sample fluid within the at least one microfluidic well while filling the at least one large-volume subwell and while filling the at least one small-volume subwell and, preferably, while filling the at least one medium-volume subwell with the sample fluid. Irrespective of this, the filling pressure can then expediently be applied for different periods of time to the sample fluid in order to fill the large-volume microchambers, preferably the at least one large-volume subwell, and the small-volume microchambers, preferably the at least one small-volume subwell, and, optionally, the medium-volume microchambers, preferably the at least one medium-volume subwell, with the sample fluid.

[0087] Irrespective of whether the filling pressure is applied at different pressure levels or at least substantially the same pressure level, the filling pressure can be applied to the sample fluid within the at least one microfluidic well by feeding gas via at least one gas flow into and / or sucking gas via at least one gas flow out of the microfluidic device and the at least one microfluidic well. In this way, the filling pressure applied to the sample fluid can be controlled very precisely with little effort, thus allowing a very reliable filling of the microchambers with the sample fluid. Expediently, the gas can be fed into the at least one microfluidic well through the well inlet opening of the microfluidic well and / or the gas can be sucked out of the at least one microfluidic well through the well outlet opening of the microfluidic well.

[0088] The at least one gas flow can for example be generated by means of at least one pipette and / or syringe. This can be particularly cost-efficient. In addition, the sample fluid can then also be introduced into the at least one microfluidic well by means of the at least one pipette and / or syringe. In this way, the amount of equipment required can be reduced. Using a pipette and / or syringe for generating the at least one gas flow can be particularly preferred if the filling pressure is applied to the sample fluid by a robotic liquid handling apparatus as these typically comprise pipettes and / or syringes anyway. Irrespective of this, the at least one pipette and / or syringe can be a piston driven pipette and / or syringe. This can be useful with respect to a precise and reliable application of the filling pressure to the sample fluid. Particularly preferably, the at least one pipette and / or syringe is a piston-driven air displacement pipette and / orsyringe. In this way, contamination of the sample fluid by the piston can be avoided when introducing the sample into the at least one microfluidic well.

[0089] Alternatively to a pipette and / or syringe, the at least one gas flow can be generated by means of at least one compressor unit. A compressor unit can be space-saving, which can be particularly useful if the filling pressure is applied to the sample fluid by an analysis apparatus as these are typically compact in design and provide only limited space.

[0090] In a preferred embodiment, the method comprises acquiring sample fluid position information, wherein the sample fluid position information represents a position of at least one sample fluid front of the sample fluid within the at least one microfluidic well, and controlling the filling pressure applied to the sample fluid within the at least one microfluidic well based on the sample fluid position information.

[0091] This allows for a very precise control of the filling pressure applied to the sample fluid. Then, expediently, the period of time for which the filling pressure is applied to the sample fluid within the at least one microfluidic well can be controlled based on the sample fluid position information. Irrespective of this, it allows in a simple and reliable manner to apply the filling pressure at the different pressure levels to the sample fluid when filling the large-volume microchambers and when filling the small-volume microchambers and, optionally, when filling the medium-volume microchambers if the large-volume microchambers and the small-volume microchambers and, optionally, the medium-volume microchambers are part of the same microfluidic well. Against this background, it can be particularly preferred if the filling pressure is controlled based on the sample fluid position information such that the filling pressure is applied at the different pressure levels to the sample fluid when filling the large-volume microchambers, preferably the at least one large-volume subwell, and when filling the smallvolume microchambers, preferably the at least one small-volume subwell, and, optionally, when filling the medium-volume microchambers, preferably the at least one medium-volume subwell, with the sample fluid. Irrespective of this, it can be expedient if the filling pressure applied to the sample fluid within the at least one microfluidic well is controlled by controlling the at least one gas flow into and / or out of the microfluidic well. Alternatively or additionally, the at least one sample fluid front which is represented by the sample fluid position information can expediently be a sample fluid flow front of the sample fluid.The sample fluid position information can simply and expediently be acquired by at least one sensor, preferably image sensor, for example camera. For the sake of simplicity, the at least one sensor acquiring the sample fluid position information can be the same as the at least one sensor acquiring the target molecule presence information. However, this does not have to be the case. Irrespective of this, the sample fluid position information can expediently represent one or more images of the sample fluid within the at least one microfluidic well, for example in the form of a video.

[0092] Expediently, the filling pressure applied to the sample fluid can be controlled based on further information in addition to the sample fluid position information, for example gas pressure information, which is indicative of a gas pressure acting on the sample fluid within the at least one microfluidic well, in particular in the inlet chamber of the at least one microfluidic well. Irrespective of this, the filling pressure applied to the sample fluid within the at least one microfluidic well can simply and expediently be controlled by an electronic control unit, for example the electronic control unit mentioned above.

[0093] In a preferred embodiment, the producing the plurality of aliquots from the sample fluid comprises fluidly separating the aliquots of the sample fluid within the microchambers of the at least one microfluidic well from each other.

[0094] In this way, exchange of sample fluid between the aliquots can be prevented after the aliquots have been introduced into the microchambers. The aliquots can easily and reliably be fluidly separated from each other by deforming, for example elastically and / or plastically deforming, the sealing layer of the microfluidic device into the channel segments of the at least one microchannel that fluidly connect the microchambers to each other. For the same reason, it can then be further preferred if the sealing layer is deformed into the channel segments by applying a compressive force to the sealing layer, preferably in a direction at least partially, in particular at least substantially, perpendicular to the sealing layer. Irrespective of the direction, the compressive force can for example be applied to the sealing layer by means of a roller and / or a clamping plate. Irrespective of how the aliquots are fluidly separated, it can be expedient if the aliquots within the microchambers of the at least one microfluidic well are separated from each other after applying the filling pressure to the sample fluid within the at least one microfluidic well. Then, it is conceivable that the application of the filling pressure to the sample fluid is stopped before the aliquots are fluidly separated from each other. However,in order to ensure that no sample fluid exits the microchambers, it can be preferred if the filling pressure on the sample fluid is maintained until at least one or more of the microchambers are fluidly separated from each other.

[0095] In a further embodiment, the method comprises introducing the sample fluid into the microfluidic device and at least one microfluidic well of the microfluidic device via the well inlet opening of the microfluidic well.

[0096] In principle, it is conceivable that the sample fluid is introduced into two or more of the microfluidic wells of the microfluidic device, in particular if the microfluidic device comprises only microfluidic wells comprising either large-volume microchambers, small-volume microchambers, or medium-volume microchambers. However, for the sake of simplicity, it is preferred if the sample fluid is introduced only into one microfluidic well comprising a plurality of the large-volume microchambers and a plurality of the small volume microchambers and, preferably, a plurality of the medium-volume microchambers. Irrespective of this, the sample fluid can expediently be introduced into the at least one microfluidic well by means of at least one pipette and / or syringe, for example by means of the at least one pipette and / or syringe by means of which also the at least one gas flow for applying the filling pressure to the sample fluid is generated. Alternatively or additionally, it can be preferred for the same reason if the sample fluid is introduced into the inlet chamber of the at least one microfluidic well via the well inlet opening of the microfluidic well.

[0097] In a further embodiment, the method comprises applying, for example gluing, a, preferably at least partially, gas permeable, protective layer to a base body of the microfluidic device forming the at least one microfluidic well for sealing the at least one well inlet opening and / or the at least one well outlet opening of the microfluidic well.

[0098] This can contribute to protect the sample fluid in the at least one microfluidic well against contamination with foreign matter and the surroundings of the microfluidic device against contamination with the sample fluid. For example, the protective layer can be applied to the base body of the microfluidic device after the filling pressure has been applied to the sample fluid within the at least one microfluidic well. However, preferably the protective layer is at least partially permeable to gas. Then, the filling pressure can be applied to the sample fluid within the at least one microfluidic well by feeding gas through the protective layer into and / or suckinggas through the protective layer out of the microfluidic well. In this way, contamination of the sample fluid and the surroundings of the microfluidic device can be prevented very reliably. Irrespective of a gas permeability of the protective layer, the protective layer can for example be applied to the base body of the microfluidic device after the sample fluid has been introduced into the at least one microfluidic well. However, it can be preferred if the sample fluid is introduced through the protective layer applied to the base body of the microfluidic device into the at least one microfluidic well, for example by means of a needle. This can also contribute to reliable protection from contamination of the sample fluid and the surroundings of the microfluidic device. Then, it can be preferred if the protective layer is configured to reseal itself after being penetrated, for example with the needle.

[0099] In a further embodiment, the method comprises thermally treating, preferably thermally cycling, the, preferably fluidly separated, aliquots, preferably within the microchambers of the at least one microfluidic well.

[0100] Then, it is expedient if the target molecule presence information is indicative, for each of the thermally treated aliquots, of whether or not the respective aliquot comprises one or more molecules of the target molecule. The target molecule presence information can then be acquired, for example only, after the thermal treatment of the aliquots has been finished.

[0101] According to a third aspect, the invention provides a system comprising the microfluidic device according to the first aspect, and a handling apparatus configured to handle the microfluidic device.

[0102] For example, the handling apparatus can be a robotic liquid handling apparatus. The robotic liquid handling apparatus can expediently comprise a plurality of pipettes and / or syringes and a robot unit configured to actuate and, preferably, move the plurality of pipettes and / or syringes. For example, the plurality of pipettes and / or syringes can be held on the robot unit. In addition, the robotic liquid handling apparatus can expediently comprise an electronic control unit configured to control the robot unit. The electronic control unit can, for example, comprise distributed components in the manner of a distributed system or be a single control device.

[0103] As an alternative to a robotic liquid handling apparatus, the handling apparatus can be an analysis apparatus configured to perform the method according to the second aspect,preferably autonomously. The analysis apparatus can expediently comprise at least one sensor, preferably image sensor, in particular camera, configured to acquire the target molecule presence information and / or a data processing unit configured to determine the amount and / or concentration of the target molecule in the sample fluid based on the acquired target molecule presence information. The data processing unit can for example be part of an electronic control unit of the analysis apparatus, which can, for example, comprise distributed components in the manner of a distributed system or can be a single control device. Alternatively or additionally to the at least one sensor and / or the data processing unit, the analysis apparatus can comprise a separating unit configured to fluidly separate the microchambers of the at least one microfluidic well from each other by deforming the sealing layer of the microfluidic device into the channel segments of the at least one microchannel that fluidly connect the microchambers to each other, preferably by applying a compressive force to the sealing layer. The separating unit can for example comprise a roller and / or a clamping plate. Irrespective of a separating unit, the analysis apparatus can comprise a thermal treatment unit configured to thermally treat, preferably thermally cycle, the aliquots within the microchambers of the at least one microfluidic well.

[0104] In a first particularly preferred embodiment of the system, the handling apparatus comprises a pressurization unit configured to apply a filling pressure to the at least one sample fluid within the at least one microfluidic well of the microfluidic device such that the sample fluid flows into at least a plurality of the large-volume microchambers and into at least a plurality of the smallvolume microchambers and, preferably, into at least a plurality of the medium-volume microchambers of the at least one microfluidic well. In this way, the microchambers can be reliably filled with the sample fluid in a short time. Preferably, the pressurization unit can be configured to apply the filling pressure to the at least one sample fluid within the at least one microfluidic well by feeding gas via at least one gas flow into and / or sucking gas via at least one gas flow out of the microfluidic device and the at least one microfluidic well. In this way, the filling pressure can be controlled very precisely with little effort, thus allowing a very reliable filling of the microchambers with the sample fluid.

[0105] For example, the pressurization unit can comprise at least one, preferably piston driven, in particular piston driven air displacement, pipette and / or syringe for generating the at least one gas flow into and / or out of the at least one microfluidic well. This can be particularly cost efficient and particularly preferred if the handling apparatus is a robotic liquid handlingapparatus as these typically comprise pipettes and / or syringes anyway. In addition, the pressurization unit can then comprise at least one sealing element for contacting the microfluidic device and sealing at least one flow path of the at least one gas flow in the contact area. This can contribute to a reliable and precise application of the filling pressure to the sample fluid. Then, it can be expedient if the at least one sealing element is made of a soft and / or elastic sealing material at least in a sealing section for contacting the microfluidic device and / or detachably connected to the at least one pipette and / or syringe. Alternatively to a pipette and / or syringe, the pressurization unit can comprise at least one compressor unit for generating the at least one gas flow into and / or out of the at least one microfluidic well. A compressor unit can be space-saving, which can be particularly useful if the handling apparatus is an analysis apparatus as these are typically compact in design and provide only limited space. Irrespective of the design of the pressurization unit, the filling pressure applied to the sample fluid within the at least one microfluidic well can expediently comprise an overpressure generated at an inlet side of the microfluidic well assigned to the well inlet opening and / or an underpressure generated at an outlet side of the microfluidic well assigned to the well outlet opening.

[0106] Alternatively or additionally, the handling apparatus can comprise an electronic control unit configured to control the filling pressure applied to the at least one sample fluid within the at least one microfluidic well. This can be expedient. Irrespective of this, the electronic control unit can expediently be configured to control the filling pressure by controlling the pressurization unit and / or the at least one gas flow into and / or out of the at least one microfluidic well.

[0107] For example, the electronic control unit can then be configured to control the filling pressure such that it is applied at different, preferably at least substantially constant, pressure levels to the sample fluid within the at least one microfluidic well when filling the large-volume microchambers with the sample fluid on the one hand and when filling the small-volume microchambers with the sample fluid on the other hand. This can be advantageous with respect to a precise and reliable filling of the microchambers with the sample fluid. For the same reason, it can then be further preferred if the electronic control unit is configured to control the filling pressure such that it is applied at a, preferably at least substantially constant, pressure level to the sample fluid within the at least one microfluidic well when filling the medium-volume microchambers with the sample fluid, which is different from the pressure levels when fillingthe large-volume microchambers and when filling the small-volume microchambers with the sample fluid. Irrespective of this, the electronic control unit can be configured to control the filling pressure applied to the at least one sample fluid within the at least one microfluidic well such that the filling pressure is applied at the different pressure levels to the sample fluid when filling the at least one large-volume subwell with the sample fluid on the one hand and when filling the at least one small-volume subwell with the sample fluid on the other hand and, preferably, when filling the at least one medium-volume subwell. A particular precise and reliable filling of the microchambers can be achieved if the electronic control unit is configured to control the filling pressure applied to the at least one sample fluid within the at least one microfluidic well such that the overpressure generated at the inlet side of the at least one microfluidic well and / or the underpressure generated at the outlet side of the at least one microfluidic well is greater when filling the large-volume microchambers, preferably the at least one large-volume subwell, with the sample fluid than when filling the small-volume microchambers, preferably the at least one small-volume subwell, with the sample fluid.

[0108] Alternatively or additionally to different pressure levels, the electronic control unit can be configured to control the filling pressure applied to the sample fluid within the at least one microfluidic well such that the filling pressure is applied at, at least substantially, the same, preferably at least substantially constant, pressure level to the sample fluid within the at least one microfluidic well while filling the large-volume microchambers and the small-volume microchambers and, preferably, the medium-volume microchambers with the sample fluid. This makes controlling the filling process very easy. For the same reason, it can be further preferred if the electronic control unit is configured to control the filling pressure such that it is applied at, at least substantially, the same, preferably at least substantially constant, pressure level to the sample fluid within the at least one microfluidic well while filling the at least one large-volume subwell and while fililng the at least one small-volume subwell and, preferably, while filling the at least one medium-volume subwell with the sample fluid.

[0109] The handling apparatus can comprise at least one sensor, preferably image sensor, in particular camera, configured to acquire sample fluid position information, which represents a position of at least one sample fluid front, in particular sample fluid flow front, of the at least one sample fluid within the at least one microfluidic well. If the handling apparatus is an analysis apparatus, for the sake of simplicity, the at least one sensor for acquiring the sample fluid position information can be the same as the at least one sensor for acquiring the targetmolecule presence information. However, this does not have to be the case. Alternatively or additionally, the sample fluid position information can expediently represent one or more images of the at least one sample fluid within the at least one microfluidic well, for example in the form of a video. Irrespective of this, the electronic control unit can be configured to control the filling pressure applied to the at least one sample fluid within the at least one microfluidic well based on the sample fluid position information. This allows a very precise control of the filling pressure applied to the at least one sample fluid. Preferably, the electronic control unit can be configured to control the filling pressure based on the sample fluid position information such that the filling pressure is applied at the different pressure levels to the at least one sample fluid when filling the large-volume microchambers with the sample fluid on the one hand and when filling the small-volume microchambers with the sample fluid on the other hand.

[0110] The abbreviation “nL” used herein stands for “nanoliter(s)”. The abbreviation “pm” used herein stands for “micrometer(s)”. And the abbreviation “mm” used herein stands for “millimeter(s)”.

[0111] It shall be understood that the microfluidic device according to the first aspect, the method according to the second aspect, and the system according to the third aspect can have similar or identical embodiments. Thus, the disclosure of a feature for one of the microfluidic device according to the first aspect, the method according to the second aspect, and the system according to the third aspect shall also be understood as disclosure of that feature and / or a corresponding feature for the other two of the microfluidic device, the method, and the system.

[0112] FIGURES

[0113] The invention is explained below with reference to a drawing showing only preferred embodiments. In the drawings,

[0114] Fig. 1 shows an embodiment of a microfluidic device according to the first aspect of the invention in a schematic perspective view,

[0115] Figs. 2A-B show details of the microfluidic device from Fig. 1 in a schematic bottom view (Fig.

[0116] 2A) and in a schematic sectional view (Fig. 2B),Figs. 3A-E show the detail of the microfluidic device from Fig. 2B, wherein the microfluidic device is shown in different situations during performing an embodiment of a method according to the second aspect of the invention,

[0117] Fig. 4 shows an embodiment of a system according to the third aspect of the invention in a schematic view,

[0118] Fig. 5 shows an alternative embodiment of a system according to the third aspect of the invention in a schematic view,

[0119] Figs. 6A-B show details of an alternative embodiment of a microfluidic device according to the first aspect of the invention in a schematic bottom view (Fig. 6A) and in a schematic sectional view (Fig. 6B), and

[0120] Figs. 7A-L show diagrams, in which the relative confidence interval over the concentration of a target molecule in a sample fluid is shown for twelve exemplary embodiments of a microfluidic device according to the first aspect.

[0121] DETAILED DESCRIPTION OF THE INVENTION

[0122] Fig. 1 shows a microfluidic device 1 according to the first aspect in a schematic perspective view. The shown and insofar preferred microfluidic device 1 is designed at least substantially plate-like and comprises a base body 2, which is also designed at least substantially plate-like. The base body 2 forms a plurality of microfluidic wells 3 each for receiving a sample fluid. In the shown and insofar preferred microfluidic device 1, the base body 2 forms 48 of the microfluidic wells 3.

[0123] In addition to the base body 2, the microfluidic device 1 comprises a sealing layer 4, which is attached, for example glued, to the bottom side of the base body 2, and a protective layer 5, which is attached, for example glued, to the top side of the base body 2. Both the sealing layer 4 and the protective layer 5 are at least substantially formed by a plastic film. In the shown and insofar preferred microfluidic device 1, the sealing layer 4 and the protective layer 5 each extend at least substantially over the entire base body 2.Figs. 2A-B show details of the microfluidic device 1 in the region of one of the microfluidic wells 3 in a schematic bottom view (Fig. 2A) and in a schematic sectional view along the sectional plane IIB-IIB shown in Fig. 2A (Fig. 2B). The other microfluidic wells 3 of the microfluidic device 1 not shown in Figs. 2A-B are designed at least substantially identical to the microfluidic well 3 shown in Fig. 2.

[0124] The microfluidic well 3 comprises a well inlet opening 6 for introducing a sample fluid into the microfluidic well 3. In the shown and insofar preferred microfluidic well 3, the well inlet opening 6 is part of an inlet chamber 7 of the microfluidic well 3 for receiving the sample fluid introduced into the microfluidic well 3 via the well inlet opening 6. The inlet chamber 7 and thus also the well inlet opening 6 are fluidly connected via a plurality of microchannels 8,9 to a plurality of microchambers 10,11 of the microfluidic well 3 each for receiving an aliquot of the sample fluid. Via the microchannels 8,9, the microchambers 10,11 are also fluidly connected to each other and to a well outlet opening 12 of the microfluidic well 3 for gas, e.g. air, displaced by the sample fluid and / or excess sample fluid to exit the microfluidic well 3. The well outlet opening 12 is part of an outlet chamber 13 of the microfluidic well 3 for receiving the displaced gas and / or the excess sample fluid, before the displaced gas and / or the excess sample fluid can exit the microfluidic well 3 via the well outlet opening 12.

[0125] The microchambers 10,11 of the microfluidic well 3 comprise a plurality of large-volume microchambers 10 and a plurality of small-volume microchambers 11. In the shown and insofar preferred microfluidic well 3, each of the large-volume microchambers 10 has a volume of approximately 7.9 nL and each of the small-volume microchambers 11 has a volume of approximately 0.019 nL (for the sake of simplicity, the volumes of the microchambers 10,11 are not shown to scale in the Figs.). Thus, the volume of each of the large-volume microchambers 10 is approximately 415 times the volume of each of the small-volume microchambers 11.

[0126] The shown and insofar preferred microfluidic well 3 comprises approximately 2250 of the large-volume microchambers 10 and approximately 22500 of the small-volume microchambers 11 (for the sake of simplicity, only a reduced number of the microchambers 10,11 are shown in the Figs.). The number of the small-volume microchambers 11 of the microfluidic well 3 is thus approximately 10 times the number of the large-volume microchambers 10 of the microfluidic well 3. Since the other not shown microfluidic wells 3 of the microfluidic device 1 are designed at least substantially identical to the shown microfluidic well 3, the number of the small-volumemicrochambers 11 of the microfluidic device 1 is also approximately 10 times the number of the large-volume microchambers 10 of the microfluidic device 1.

[0127] The microfluidic well 3 comprises a large-volume subwell 14 which comprises the large-volume microchambers 10 of the microfluidic well 3 and the parallel microchannels 8 arranged in the region of the large-volume microchambers 10, of which there are two in the embodiment shown. In addition, the microfluidic well 3 comprises a small-volume subwell 15 which comprises the small-volume microchambers 11 of the microfluidic well 3 and the parallel microchannels 9 arranged in the region of the small-volume microchambers 11, of which there are six in the embodiment shown. The large-volume subwell 14 is fluidly connected to the outlet chamber 13 and the well outlet opening 12 via the small-volume subwell 15. And the smallvolume subwell 15 is fluidly connected to the inlet chamber 7 and the well inlet opening 6 via the large-volume subwell 14.

[0128] The large-volume subwell 14 is fluidly connected to the small-volume subwell 15 via channel branches 16, of which there are two in the embodiment shown. Via the channel branches 16, in each case, one of the microchannels 8 of the large-volume subwell 14 is fluidly connected to three of the microchannels 9 of the small-volume subwell 15. In this way, the fluid flows of the sample fluid flowing through the microchannels 8 of the large-volume subwell 14 in the direction of the outlet chamber 13 and the well outlet opening 12 can each be divided into three fluid flows, thus in total six fluid flows, flowing through the small-volume subwell 15.

[0129] In the shown and insofar preferred embodiment, the sealing layer 4 attached to the bottom side of the base body 2 of the microfluidic device 1 extends over the entire microfluidic well 3 (and also over the other microfluidic wells 3 of the microfluidic device 1 that are not shown in Figs. 2A-B). However, the sealing layer 4 is transparent so that the base body 2 and the microfluidic well 3 are visible through the sealing layer 4 (cf. Fig. 2A).

[0130] The protective layer 5 attached to the top side of the base body 2 of the microfluidic device 1 seals the well inlet opening 6 and the well outlet opening 12 of the microfluidic well 3 (and also the well inlet openings 6 and the well outlet openings 12 of the other microfluidic wells 3 of the microfluidic device 1 that are not shown in Figs. 2A-B). In the shown and insofar preferred embodiment, the protective layer 5 also extends entirely over all the microfluidic wells 3 of the microfluidic device 1.Figs. 3A-E show the detail of the microfluidic device 1 from Fig. 2B, wherein the microfluidic device 1 is shown in different situations during performing a method according to the second aspect.

[0131] In Fig. 3A, a sample fluid SF has been introduced via the well inlet opening 6 into the microfluidic well 3, namely into the inlet chamber 7 of the microfluidic well 3.

[0132] In the shown embodiment, the sample fluid SF has been introduced into the inlet chamber 7 through the protective layer 5 sealing the well inlet opening 6, for example by means of a needle, e.g. an injection needle, or the like. The protective layer 5 is designed such that it reseals itself after having been penetrated, for example with the needle, in order to introduce the sample fluid SF into the inlet chamber 7. In this way, it can be ensured that the protective layer 5 reliably protects the sample fluid SF and the surroundings of the microfluidic device 1 against contamination after the introduction of the sample fluid SF into the inlet chamber 7.

[0133] Alternatively, the protective layer 5 could also be attached to the base body 2 of the microfluidic device 1, after the sample fluid SF has been introduced into the microfluidic well 3. This can be easier and reduce the requirements for the protective layer 5. However, introducing the sample fluid SF through the protective layer 5 into the microfluidic well 3 allows a very reliable protection against contamination of the sample fluid SF and the surroundings of the microfluidic device 1.

[0134] In order to force the sample fluid SF from the inlet chamber 7 into the large-volume subwell 14 and the small-volume subwell 15, in a next step, a filling pressure is applied to the sample fluid SF in the inlet chamber 7.

[0135] In Fig. 3B, the filling pressure FP is being applied to the sample fluid SF within the inlet chamber 7. The filling pressure FP is applied to the sample fluid SF by feeding gas via a gas flow GF into the microfluidic device 1 and the inlet chamber 7. In the shown and insofar preferred embodiment, the gas is fed through the protective layer 5, which is permeable to gas in the present embodiment, into the inlet chamber 7. However, alternatively, the protective layer 5 could also be attached to the base body 2 of the microfluidic device 1 , after the filling pressure FP has been applied to the sample fluid SF. Then, the protective layer 5 does not necessarilyhave to be permeable to gas. However, with respect to a reliable protection against contamination of the sample fluid SF and the surroundings of the microfluidic device 1, it can be preferred if the gas is fed through the protective layer 5 into the inlet chamber 7.

[0136] In the shown and insofar preferred embodiment, the filling pressure FP is an overpressure generated at the inlet side of the microfluidic well 3 assigned to the well inlet opening 6. However, although this can be preferred, the filling pressure FP could alternatively or additionally also be or comprise an underpressure generated at the outlet side of the microfluidic well 3 assigned to the well outlet opening 6 by sucking gas via a gas flow out of the outlet chamber 13 of the microfluidic well 3.

[0137] As a result of the applied filing pressure FP, the sample fluid SF flows from the inlet chamber 7 via the microchannels 8,9 into all the large-volume microchambers 10 of the large-volume subwell 14 and substantially all the small-volume microchambers 11 of the small-volume subwell 15. In the course of this, the filling pressure FP is applied at different pressure levels to the sample fluid SF when filing the large-volume subwell 14 with the sample fluid SF on the one hand and when filling the small-volume subwell 15 with the sample fluid SF on the other hand. In order to control the filling pressure FP applied to the sample fluid SF, sample fluid position information representing a position of at least one sample fluid front SFF of the sample fluid SF within the microfluidic well 3 is acquired. For example, the sample fluid position information can be acquired by a camera. Then, the sample fluid position information can represent one or more images (e.g. in the form of a video) of the sample fluid SF within the microfluidic well 3 acquired through the transparent sealing layer 4 of the microfluidic device 1. Based on the acquired sample fluid position information, the gas flow GF into the inlet chamber 7 of the microfluidic well 3 and thus the filling pressure FP applied to the sample fluid SF is controlled, for example by an electronic control unit, such that the filling pressure FP is applied at a higher pressure level when filling the large-volume subwell 14 with the sample fluid SF than when filling the small-volume subwell 15 with the sample fluid SF. In addition, the gas flow GF into the inlet chamber 7 can be controlled based on the acquired sample fluid position information such that the gas flow GF into the inlet chamber 7 is stopped when the sample fluid front SFF reaches a predetermined position.

[0138] In Fig. 30, all the large-volume microchambers 10 and substantially all the small-volume microchambers 11 have been filled with the sample fluid SF as a result of the application ofthe filling pressure FP to the sample fluid SF. Only some of the small-volume microchambers 11 in the region adjacent to the outlet chamber 13 have not been filled with the sample fluid SF as a result of the applied filling pressure FP. While the gas flow GF into the inlet chamber 7 has been stopped, the filling pressure FP applied to the sample fluid SF is maintained to prevent sample fluid SF from escaping the microchambers 10,11 again, for example due to capillary forces.

[0139] In order to fill also the remaining, sample fluid-free small-volume microchambers 11 with the sample fluid SF and to fluidly separate the microchambers 10,11 from each other, in a next step, a compressive force is applied to the sealing layer 4 in the direction of the base body 2.

[0140] In Fig. 3D, the compressive force CF is being applied to the sealing layer 4, for example by means of a roller which is rolled over the sealing layer 4 from the section of the sealing layer 4 adjacent to the inlet chamber 7 into the direction of the outlet chamber 13. As a result of the applied compressive force CF, the sealing layer 4 is being deformed into the microchannels 8,9. In this way, the sample fluid SF is forced from the microchannels 8,9 into the remaining sample fluid-free small-volume microchambers 11 and the excess sample fluid SF into the outlet chamber 13, and the microchambers 10,11 are fluidly separated from each other.

[0141] In Fig. 3E, all the microchambers 10,11 have been fluidly separated from each other. In this way, a plurality of large-volume aliquots LVA contained in the large-volume microchambers 10 and a plurality of small-volume aliquots SVA contained in the small-volume microchambers 11 have been produced. In the shown and insofar preferred embodiment, approximately 2250 of the large-volume aliquots LVA and approximately 22500 of the small-volume aliquots SVA have been produced, and the volume of each of the large-volume aliquots LVA is approximately 7.9 nL and the volume of each of the small-volume aliquots SVA is approximately 0.019 nL so that the volume of each of the large-volume aliquots LVA is approximately 415 times the volume of each of the small-volume aliquots SVA.

[0142] In a next step, the fluidly separated aliquots LVA, SVA are thermally treated, preferably thermally cycled, within the microchambers 10,11 of the microfluidic well 3, for example by means of a thermal treatment unit, e.g. of an analysis apparatus. After the thermal treatment, target molecule presence information is acquired, which is indicative, for each of the aliquots LVA, SVA, of whether or not the respective aliquot LVA, SVA comprises one or more moleculesof a target molecule. The target molecule presence information can for example be acquired by a camera, e.g. of the analysis apparatus. Then, the target molecule presence information can represent one or more images of the aliquots LVA.SVA within the microchambers 10,11 acquired through the transparent sealing layer 4. Before and / or while the target molecule presence information is acquired, the aliquots LVA.SVA can be illuminated through the transparent sealing layer 4 with excitation light, for example with one or more wavelengths from the ultraviolet range. Then, the target molecule presence information can be indicative, for each of the aliquots LVA.SVA, of whether or not the respective aliquot LVA.SVA fluoresces, wherein it can indicate the presence of one or more molecules of the target molecule in the respective aliquot LVA.SVA if the aliquot LVA.SVA fluoresces. Finally, the amount and / or concentration of the target molecule in the sample fluid SF is determined based on the acquired target molecule presence information, for example by a data processing unit of the analysis apparatus.

[0143] Fig. 4 shows a system 17 according to the third aspect in a schematic view. The system 17 comprises the microfluidic device 1 according to the first aspect shown in Figs. 1-3E and a handling apparatus 18 in the form of an analysis apparatus 18 configured to perform a method according to the second aspect.

[0144] The analysis apparatus 18 comprises a holding device 19 for holding the microfluidic device 1 in the inside of the analysis apparatus 18 during handling of the microfluidic device 1 by the analysis apparatus 18. The holding device 19 can for example comprise a pair of rails for moving the microfluidic device 1 from a position outside the analysis apparatus 18 into a position inside the analysis apparatus 18 and back.

[0145] Before the microfluidic device 1 is inserted into the analysis apparatus 18, sample fluids SF can have been introduced, e.g. manually, into the microfluidic device 1 and the microfluidic wells 3. For example, the sample fluids SF can have been introduced into the microfluidic device 1 and the microfluidic wells 3, before the protective layer 5 was attached to the base body 2 of the microfluidic device 1. Alternatively, the sample fluids SF can also have been introduced through the protective layer 5 into the microfluidic device 1 and the microfluidic wells 3, for example by means of one or more needles.In order to apply a filling pressure FP to the sample fluids SF within the microfluidic wells 3 of the microfluidic device 1 such that the sample fluids SF flow into substantially all the microchambers 10,11 of the respective microfluidic well 3, the analysis apparatus 18 comprises a pressurization unit 20. The pressurization unit 20 is configured to apply the filling pressure FP to the sample fluids SF by feeding gas via a plurality of gas flows GF through the protective layer 5 into the microfluidic device 1 and the microfluidic wells 3. For generating the gas flows GF and guiding them into the microfluidic wells 3, the pressurization unit 20 comprises a compressor unit 21 for generating a gas flow GF and a gas guiding structure 22 for splitting up the gas flow generated by the compressor unit 21 into a plurality of gas flows GF and guiding these gas flows GF into the microfluidic wells 3 of the microfluidic device 1.

[0146] For controlling the pressurization unit 20, the analysis apparatus 18 comprises an electronic control unit 23. The electronic control unit 23 is configured to control the pressurization unit 20 such that the filling pressure FP applied to the sample fluids SF within the microfluidic wells 3 is applied at different pressure levels to the respective sample fluid SF when filling the large-volume microchambers 10 of the respective microfluidic well 3 with the sample fluid SF on the one hand and when filling the small-volume microchambers 11 of the respective microfluidic well 3 with the sample fluid SF on the other hand. To this end, the electronic control unit 23 receives sample fluid position information from an image sensor 24, for example in the form of a camera, of the analysis apparatus 18, which is indicative of positions of sample fluid fronts SFF of the sample fluids SF within the microfluidic wells 3 and represents one or more images, for example in the form of a video, of the sample fluids SF within the microfluidic wells 3 captured through the transparent sealing layer 4 of the microfluidic device 1. The electronic control unit 23 can then evaluate the received sample fluid position information, for example by means of an image recognition software, and control the pressurization unit 20 and thus the gas flows GF into the microfluidic wells 3 based on the results of the evaluation of the sample fluid position information. Expediently, the electronic control unit 23 can be configured to control the different gas flows GF into the microfluidic wells 3 independently of each other, for example by means of one or more valves.

[0147] Further, the analysis apparatus 18 comprises a separating unit 25 for applying a compressive force CF to the sealing layer 4 of the microfluidic device 1 such that the sealing layer 4 is deformed into the microchannels 8,9 of the microfluidic wells 3, thus fluidly separating the microchambers 10,11 of the microfluidic wells 3 and the aliquots LVA.SVA contained thereinfrom each other. The shown and insofar preferred separating unit 25 comprises a roller 26 for applying the compressive force CF to the sealing layer 4, which can be rolled over the sealing layer 4.

[0148] In order to thermally treat, in particular thermally cycle, the fluidly separated aliquots LVA.SVA of the different sample fluids SF within the microchambers 10,11 of the respective microfluidic well 3, the analysis apparatus 18 comprises a thermal treatment unit 27. The thermal treatment unit 27 can for example comprise one or more heating elements 28 for heating the aliquots LVA.SVA within the microfluidic wells 3 of the microfluidic device 1.

[0149] After the thermal treatment, the electronic control unit 23 can determine an amount and / or a concentration of a target molecule in each of the sample fluids SF. For this purpose, the analysis apparatus 18 comprises an excitation light source 29 configured to illuminate the aliquots LVA.SVA in the microfluidic wells 3 through the transparent sealing layer 4 of the microfluidic device 1 with excitation light, for example having one or more wavelengths in the ultraviolet range. As a result of the illumination with the excitation light, the aliquots LVA.SVA containing at least one of the target molecule can fluoresce. The image sensor 24 can then acquire target molecule presence information, which represents one or more images of the aliquots LVA.SVA within the microchambers 10,11 of the microfluidic wells 3 and is indicative, for each of the aliquots LVA.SVA, of whether or not the respective aliquot LVA.SVA fluoresces, and transmit the target molecule presence information to the electronic control unit 23. Based on the target molecule presence information, the electronic control unit 23 can then determine, for each of the sample fluids SF in the microfluidic wells 3, the amount and / or concentration of the target molecule in the respective sample fluid SF.

[0150] Fig. 5 shows an alternative system 30 according to the third aspect in a schematic view. The system 30 comprises a microfluidic device 31 and a handling apparatus 32 in the form of a robotic liquid handling apparatus 32. The shown microfluidic device 31 is similar to the microfluidic device 1 shown in Figs. 1-4. The only difference is that the shown microfluidic device 31 does not comprise the gas-permeable protective layer 5 of the microfluidic device 1 shown in Figs. 1-4. For the rest, the design of the shown microfluidic device 31 is the same as that of the microfluidic device 1 shown in Figs. 1-4.The robotic liquid handling apparatus 32 comprises a pressurization unit 33 which comprises a plurality of pipettes 34 and / or syringes 34 in the form of piston-driven air displacement pipettes 34 and / or syringes 34. In addition, the pressurization unit 33 comprises a plurality of sealing elements 35, wherein some of the sealing elements 35 are detachably connected to the free ends of the pipettes 34 and / or syringes 34. Each of the sealing elements 35 comprises a soft and / or elastic sealing section 36 and a connecting section 37 for connecting the respective sealing element 35 to one of the pipettes 34 and / or syringes 34. The plurality of pipettes 34 and / or syringes 34 are held on a robot unit 38 of the robotic liquid handling apparatus 32. The robot unit 38 is configured to actuate and move the pipettes 34 and / or syringes 34, for example in all three spatial directions.

[0151] The robotic liquid handling apparatus 32 further comprises a workdeck 39. On the workdeck 39, a holding device 40 is provided for holding the microfluidic device 31. In addition, a storge unit 41 , for example in the form of a rack or the like, is provided on the workdeck 39 for holding the sealing elements 35 available when they are not in use. The storage unit 41 comprises a plurality of receptacles 42 for the sealing elements 35. Further, a layer application unit 43 is provided on the workdeck 39 for applying a protective layer to the top side of the base body 2 of the microfluidic device 31 in order to seal the well inlet openings 6 and well outlet openings 12 of the microfluidic wells 3 of the microfluidic device 31. The layer application unit 43 comprises a transport device 44, for example in the form of a movable slide, on which the microfluidic device 31 can be placed and by means of which the microfluidic device 31 can be moved into the layer application unit 43 for the application of the protective layer. Such layer application units are known in the prior art.

[0152] Further, the robotic liquid handling apparatus 32 comprises an electronic control unit 45, which is configured to control the robot unit 38 and the layer application unit 43.

[0153] By means of the pipettes 34 and / or syringes 34 sample fluids SF can be introduced into the microfluidic wells 3 of the microfluidic device 31. For this purpose, the sealing elements 35 can be disconnected from the pipettes 34 and / or syringes 34 and stored in the storage unit 41. Then, for example, pipette tips can be connected to the pipettes 34 and / or syringes 34 to pipette the sample fluids SF into the microfluidic wells 3.After the sample fluids SF have been introduced into the microfluidic device 31 and the pipette tips have been disconnected from the pipettes 34 and / or syringes 34, one of the sealing elements 35 can be connected to each of the pipettes 34 and / or syringes 34 at the storage unit 41. Thereafter, the robot unit 38 can move the pipettes 34 and / or syringes 34 together with the connected sealing elements 35 from the storage unit 41 to the microfluidic device 31. During this movement, a predetermined volume of gas, in particular air, can be sucked through the respective sealing element 35 into each of the pipettes 34 and / or syringes 34 by actuating the pistons 46 of the pipettes 34 and / or syringes 34. Then, the robot unit 38 can press the sealing elements 35 connected to the pipettes 34 and / or syringes 34 with their respective sealing section 36 against the top side of the base body 2 of the microfluidic device 31 such that each of the sealing sections 36 contacts the base body 2 circumferentially around the well inlet opening 6 of a respective one of the microfluidic wells 3. Thereafter, the pistons 46 of the pipettes 34 and / or syringes 34 can be pushed in the direction of the respective sealing element 35, thereby feeding gas via a gas flow GF into the well inlet opening 6 of the respective microfluidic well 3. In this way, a filling pressure FP can be applied to the sample fluids SF within the microfluidic wells 3, as a result of which the sample fluids SF flow into at least substantially all the microchambers 10,11 of the respective microfluidic well 3.

[0154] The electronic control unit 45 is configured to control the robot unit 38 and thus the pipettes 34 and / or syringes 34 such that the filling pressure FP is applied at a higher pressure level to the sample fluids SF within the microfluidic wells 3 when filling the large-volume microchambers 10 of the respective microfluidic well 3 with the respective sample fluid SF than when filling the small-volume microchambers 11 of the respective microfluidic well 3 with the respective sample fluid SF. To this end, the electronic control unit 45 can receive from a sensor 46, for example image sensor, sample fluid position information indicative of positions of sample fluid fronts SFF of the sample fluids SF within the microfluidic wells 3, and control the robot unit 38 and thus the pipettes 34 and / or syringes 34 based on the received sample fluid position information. The sensor 46 can for example be provided in the holding device 40 or, alternatively, in the workdeck 39 below the holding device 40.

[0155] After the filling pressure FP has been applied to the sample fluids SF within the microfluidic wells 3, the robot unit 38 can move the microfluidic device 31 from the holding device 40 onto the transport device 44 of the layer application unit 43, for example by means of a gripper device. The transport device 44 can then move the microfluidic device 31 into the layerapplication unit 43, where a protective layer, for example in the form of a gas-impermeable protective layer, can be applied by the layer application unit 43 to the upper side of the base body 2 of the microfluidic device 31 in order to seal the well inlet openings 6 and the well outlet openings 12 of the microfluidic wells 3.

[0156] In addition to the application of the filling pressure FP to the sample fluids SF, the electronic control unit 45 can also control the introduction of the sample fluids SF into the microfluidic wells 3 by controlling the robot unit 38 and / or the application of the protective layer to the base body 2 of the microfluidic device 31 by controlling the layer application unit 43.

[0157] After the protective layer has been applied to the base body 2 of the microfluidic device 31 , the microfluidic device 31 can be transferred, for example manually, from the robotic liquid handling apparatus 32 into an analysis apparatus, for example an analysis apparatus at least similar to the one shown in Fig. 4. The analysis apparatus can then fluidly separate the aliquots LVA.SVA in the microchambers 10,11 of the microfluidic wells 3, thermally treat the fluidly separated aliquots LVA.SVA, and determine the amount and / or concentration of a target molecule in each of the sample fluids SF.

[0158] Figs. 6A-B show details of an alternative embodiment of a microfluidic device T according to the first aspect in a region of one of the microfluidic wells 3’ of the microfluidic device in a schematic top view and a schematic sectional view. The shown embodiment of the microfluidic device 1 is similar to the embodiment in Figs. 1-3E. Therefore, identical and similar components also have the same reference signs.

[0159] The main difference between the shown embodiment of the microfluidic device 1 and the embodiment in Figs. 1-3E is that the microfluidic wells 3 of the shown microfluidic device 1 comprise medium-volume microchambers 48 in addition to the large- and small-volume microchambers 10,11. The volume of each of the medium-volume microchambers 48 is smaller than the volume of each of the large-volume microchambers 10 and greater than the volume of each of the small-volume microchambers 11.

[0160] The microfluidic well 3 comprises a medium-volume subwell 49 which comprises the mediumvolume microchambers 48 of the microfluidic well 3. In the shown and insofar preferred microfluidic well 3, the medium-volume subwell 49 is fluidly connected to the outlet chamber13 and the well outlet opening 12 via the small-volume subwell 15 and to the inlet chamber 7 and the well inlet opening 6 via the large-volume subwell 14. The medium-volume subwell 49 is fluidly connected to the small-volume subwell 15 via the channel branches 16.

[0161] Figs. 7A-L show diagrams, in which the relative confidence interval in % with a confidence level of 95 % over the concentration of a target molecule in a sample fluid in cp / pl is shown for twelve exemplary embodiments, embodiments A-L, of a microfluidic device according to the first aspect. The diagram in Fig. 7A pertains to embodiment A, the diagram in Fig. 7B to embodiment B, the diagram in Fig. 7C to embodiment C, the diagram in Fig. 7D to embodiment D, the diagram in Fig. 7E to embodiment E, the diagram in Fig. 7F to embodiment F, the diagram in Fig. 7G to embodiment G, the diagram in Fig. 7H to embodiment H, the diagram in Fig. 7I to embodiment I, the diagram in Fig. 7J to embodiment J, the diagram in Fig. 7K to embodiment K, and the diagram in Fig. 7L to embodiment L.

[0162] The relevant specifications of the embodiments A-L are shown in the following tables:

[0163]

[0164]

[0165] In this context of dPCR technology, A is defined as the natural log of the quotient of the number of negative droplets and the number of total accepted droplets.

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Each of the diagrams shows a continuous curve C1, a dotted curve C2, and, optionally, a dashed curve C3. The continuous curve C1 shows in each case the relative confidence interval over the concentration of the target molecule in the sample fluid if only the large-volume microchambers of one of the microfluidic wells of the respective microfluidic device are used for determining the concentration of the target molecule in the sample fluid. The dotted curve C2 shows in each case the relative confidence interval over the concentration of the target molecule in the sample fluid if only the small-volume microchambers of one of the microfluidic wells of the respective microfluidic device are used for determining the concentration of the target molecule in the sample fluid. And the dashed curve C3 shows in each case the relative confidence interval over the concentration of the target molecule in the sample fluid if only the medium-volume microchambers of one of the microfluidic wells of the respective microfluidicdevice are used for determining the concentration of the target molecule in the sample fluid. The relative confidence interval when using both the large-volume microchambers and the small-volume microchambers and, optionally, the medium-volume microchambers of the respective microfluidic well for the determination of the concentration of the target molecule in the sample fluid results in each case from the synopsis of the two or three curves C1 , C2, and optionally C3.

[0172] As can be seen from the diagrams, while the embodiment A (Fig. 7A) provides very low relative confidence intervals at concentrations of 100 cp / pL to 10000 cp / pL, the embodiments B-E and l-L (Figs.7B-E and l-L) provide low relative confidence intervals over a significantly wider range of concentrations than the embodiment A. And the embodiments B-E and l-L do also provide a significantly larger dynamic range than embodiment A and in particular a greater maximum detectable concentration, wherein embodiments E, K and L provide the largest dynamic range and at the same time the smallest minimum detectable concentration and the highest maximum detectable concentration (cf. table above). Further, Figs. 7F-H illustrating embodiments F, G and H demonstrate an effect of the medium-volume microchambers allowing for particularly low relative confidence intervals for concentrations between those particularly effectively addressed by the large-volume microchambers, on the one hand and the small-volume microchambers, on the other hand.

[0173] The data that the diagrams are based on as well as the lowest and highest detectable concentration and the dynamic range indicated in the tables above were simulated by means of a simulation tool.

[0174] REFERENCE SIGNS

[0175] 1 microfluidic device

[0176] 2 base body

[0177] 3 microfluidic well

[0178] 4 sealing layer

[0179] 5 protective layer

[0180] 6 well inlet opening

[0181] 7 inlet chamber

[0182] 8 microchannel9 microchannel

[0183] 10 large-volume microchamber 11 small-volume microchamber 12 well outlet opening

[0184] 13 outlet chamber

[0185] 14 large-volume subwell

[0186] 15 small-volume subwell

[0187] 16 channel branch

[0188] 17 system

[0189] 18 analysis apparatus

[0190] 19 holding device

[0191] 20 pressurization unit

[0192] 21 compressor unit

[0193] 22 gas guiding structure

[0194] 23 electronic control unit

[0195] 24 image sensor

[0196] 25 separating unit

[0197] 26 roller

[0198] 27 thermal treatment unit

[0199] 28 heating element

[0200] 29 excitation light source

[0201] 30 system

[0202] 31 microfluidic device

[0203] 32 robotic liquid handling apparatus 33 pressurization unit

[0204] 34 pipette and / or syringe

[0205] 35 sealing element

[0206] 36 sealing section

[0207] 37 connecting section

[0208] 38 robot unit

[0209] 39 workdeck

[0210] 40 holding device

[0211] 41 storage unit

[0212] 42 receptacle43 layer application unit

[0213] 44 transport device

[0214] 45 electronic control unit

[0215] 46 sensor

[0216] 47 piston

[0217] 48 medium-volume microchambers 49 medium-volume subwell

[0218] CF compressive force

[0219] FP filling pressure

[0220] GF gas flow

[0221] LVA large-volume aliquot

[0222] SF sample fluid

[0223] SFF sample fluid front

[0224] SVA small-volume aliquot

[0225] C1 continuous curve

[0226] 02 dotted curve

[0227] 03 dashed curve

Claims

1. CLAIMS1. A microfluidic device (1,31) for handling at least one sample fluid (SF) and producing a plurality of aliquots (LVA.SVA) from the sample fluid (SF), the microfluidic device (1,31) comprisingat least one microfluidic well (3) for receiving the at least one sample fluid (SF), - wherein the microfluidic well (3) comprises at least one well inlet opening (6) for introducing the sample fluid (SF) into the microfluidic well (3), a plurality of microchambers (10,11) each for receiving an aliquot (LVA.SVA) of the plurality of aliquots (LVA.SVA) of the sample fluid (SF), and at least one well outlet opening (12) for gas displaced by the sample fluid (SF) and / or excess sample fluid (SF) to exit the microfluidic well (3), wherein the microchambers (10,11) of the microfluidic well (3) are fluidly connected to each other and to the well inlet opening (6) and the well outlet opening (12) of the microfluidic well (3) via at least one microchannel (8,9) of the microfluidic well (3),- wherein the microchambers (10,11) of the at least one microfluidic well (3) comprise a plurality of large-volume microchambers (10) and a plurality of small-volume microchambers (11), andwherein the volume of each of the large-volume microchambers (10) is at least 1.5 times and at most 100000 times the volume of each of the small-volume microchambers (11).

2. The microfluidic device according to claim 1, wherein the volume of each of the large- volume microchambers (10) is at least 100 times, at least 150 times, at least 180 times, at least 250 times, at least 350 times, at least 400 times, at least 500 times, at least 750 times, at least 850 times, or at least 900 times, and / or at most 10000 times, at most 5000 times, at most 2000 times, at most 1500 times, at most 1200 times, at most 1000 times, at most 800 times, at most 600 times, at most 500 times, or at most 450 times the volume of each of the small-volume microchambers (11).

3. The microfluidic device according to claim 1 and / or 2, wherein the volume of each of the large-volume microchambers (10) is at least 0.5 nL, at least 1 nL, at least 1.5 nL, at least 2 nL, at least 2.3 nL, at least 5 nL, at least 7 nL, at least 8 nL, at least 8.5 nL, or at least- 60 -8.8 nL, and / or at most 50 nL, at most 20 nL, at most 15 nL, at most 12 nL, at most 10 nL, at most 9.5 nL, at most 9.3 nL, at most 7 nL, at most 5 nL, or at most 3 nL, and / or wherein the volume of each of the small-volume microchambers (11) is at most 0.8 nL, at most 0.5 nL, at most 0.4 nL, at most 0.1 nL, at most 0.05 nL, at most 0.02 nL, at most 0.015 nL, at most 0.012 nL, at most 0.01 nL, or at most 0.008 nL, and / or at least 0.001 nL, at least 0.003 nL, at least 0.005 nL, at least 0.007 nL, or at least 0.008 nL4. The microfluidic device according to one or more of claims 1 to 3, wherein the microchambers (10,11) of the at least one microfluidic well (3) comprise a plurality of medium-volume microchambers, wherein the volume of each of the medium-volume microchambers is smaller than the volume of each of the large-volume microchambers (10) and greater than the volume of each of the small-volume microchambers (11), and wherein, preferably, the volume of each of the medium-volume microchambers is at least 0.01 nL, at least 0.1 nL, at least 0.3 nL, at least 0.4 nL, at least 1 nL, at least 1.5 nL, at least 1.8 nL, at least 2.3 nL, or at least 2.5 nL, and / or at most 10 nL, at most 5 nL, at most 3.5 nL, at most 2.5 nL, at most 2.2 nL, at most 1.5 nL, at most 1 nL, or at most 0.8 nL.

5. The microfluidic device according to claim 4, wherein the volume of each of the large- volume microchambers is at least 1.5 times, at least 2.5 times, at least 3 times, at least 5 times, at least 10 times, or at least 12 times, and / or at most 50 times, at most 30 times, at most 20 times, at most 15 times, at most 10 times, or at most 7 times the volume of each of the medium-volume microchambers, and / or wherein the volume of each of the medium-volume microchambers is at least 10 times, at least 30 times, at least 50 times, at least 100 times, at least 150 times, at least 180 times, at least 200 times, at least 250 times, or at least 280 times, and / or at most 500 times, at most 400 times, at most 350 times, at most 320 times, at most 250 times, at most 230 times, at most 150 times, at most 100 times, or at most 80 times the volume of each of the small-volume microchambers.

6. The microfluidic device according to one or more of claims 1 to 5, wherein the number of the small-volume microchambers (11) of the microfluidic device (1 ,31) is greater than, preferably at least 1.5 times, at least 1.8 times, at least 2 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 50 times, or at least 70 times, and / or- 61 -at most 200 times, at most 100 times, at most 50 times, at most 30 times, at most 25 times, at most 15 times, at most 10 times, at most 8 times, or at most 6 times, the number of the large-volume microchambers (10) of the microfluidic device (1,31), and / or wherein the number of the small-volume microchambers (11) of the microfluidic device (1,31) is greater than, preferably at least 2 times, in particular at least 3 times, particularly preferably at least 4 times, and / or at most 30 times, in particular at most 15 times, particularly preferably at most 10 times, the number of the medium-volume microchambers of the microfluidic device, and / or wherein the number of the large-volume microchambers of the microfluidic device is greater than, preferably at least 1.1 times, in particular at least 1.3 times, particularly preferably at least 1.4 times, and / or at most 10 times, in particular at most 5 times, particularly preferably at most 3 times, the number of the medium-volume microchambers of the microfluidic device.

7. The microfluidic device according to one or more of claims 1 to 6, wherein the large- volume microchambers (10) have at least substantially the same volume and / or the small-volume microchambers (11) have at least substantially the same volume and / or the medium-volume microchambers have at least substantially the same volume, and / or wherein the microfluidic device (1,31) comprises a plurality of, preferably at least substantially identically designed, microfluidic wells (3), preferably at least 5, at least 20, at least 40, or at least 80, and / or at most 384, at most 120, at most 60, at most 30, or at most 15microfluidic wells (3).

8. The microfluidic device according to one or more of claims 1 to 7, wherein the microfluidic well (3), preferably each of the microfluidic wells (3), comprises a plurality of the large- volume microchambers (10) and a plurality of the small-volume microchambers (11) and, preferably, a plurality of the medium-volume microchambers, and wherein, preferably, the microfluidic well (3), in particular each of the microfluidic wells (3), comprises at least 500, preferably at least 800, in particular at least 1000, of the large-volume microchambers, of the small-volume microchambers, and / or of the medium-volume microchambers.

9. The microfluidic device according to claim 8, wherein the microfluidic well, preferably each of the microfluidic wells, comprises at least at least 1500, at least 1800, at least 1900, at least 2300, at least 2400, at least 2800, or at least 2900, and / or at most 10000,- 62 -at most 5000, at most 3500, at most 3200, at most 2800, at most 2600, at most 2300, or at most 2100 of the large-volume microchambers (10), and / or at least 2000, at least 4000, at least 4500, at least 8000, at least 9000, at least 13000, at least 14000, at least 25000, at least 40000, at least 45000, at least 150000, at least 180000, or at least 200000 and / or at most 300000, at most 250000, at most 220000, at most 100000, at most 60000, at most 55000, at most 30000, at most 20000, at most 17000, at most 16000, at most 12000, at most 11000, at most 7000, at most 6000, or at most 5500 of the small-volume microchambers (11), and / or at most 5000, preferably at most 3000, in particular at most 2000, or at most 1500, of the medium-volume microchambers.

10. The microfluidic device according to claim 8 and / or 9, wherein the microfluidic well (3), preferably each of the microfluidic wells (3), comprises at least one large-volume subwell (14) comprising the large-volume microchambers (10) of the microfluidic well (3) and at least one small-volume subwell (15) comprising the small-volume microchambers (11) of the microfluidic well (3) and, preferably, at least one medium-volume subwell comprising the medium-volume microchambers, and wherein, preferably, the at least one large-volume subwell (14) of the microfluidic well (3), in particular of each of the microfluidic wells (3), is fluidly connected to the well outlet opening (12) or the well inlet opening (6) of the microfluidic well (3) via the at least one small-volume subwell (15) and / or the at least one medium-volume subwell of the microfluidic well (3) and / or the at least one small-volume subwell (15) of the microfluidic well (3), in particular of each of the microfluidic wells (3), is fluidly connected to the well inlet opening (6) or the well outlet opening (12) of the microfluidic well (3) via the at least one large-volume subwell (14) and / or the at least one medium-volume subwell of the microfluidic well (3).

11. The microfluidic device according to one or more of claims 1 to 10, wherein the microfluidic device (1,31) comprises a sealing layer (4) for fluidly separating the microchambers (10,11) of the at least one microfluidic well (3) from each other by sealing channel segments of the at least one microchannel (8,9) fluidly connecting the microchambers (10,11) to each other, preferably by deformation into the channel segments, in particular upon application of a compressive force (CF) to the sealing layer (4), and wherein, preferably, the sealing layer (4) comprises a film, in particular a plastic film, and / or is attached, in particular glued, to a base body (2) of the microfluidic device (1,31) forming the at least one microfluidic well (3).- 63 -12. The microfluidic device according to one or more of claims 1 to 11, wherein the microfluidic device (1,31) comprises a, preferably at least partially gas permeable, protective layer (5) sealing the at least one well inlet opening (6) and / or the at least one well outlet opening (12), and wherein, preferably, the protective layer (5) comprises a film, in particular a plastic film, and / or is attached, in particular glued, to a base body (2) of the microfluidic device (1,31) forming the at least one microfluidic well (3).

13. A method, preferably digital PCR method, for determining an amount and / or a concentration of a target molecule, preferably nucleic acid molecule, in a sample fluid (SF), preferably using the microfluidic device (1,31) according to one or more of claims 1 to 12, the method comprisingproducing a plurality of aliquots (LVA.SVA) from the sample fluid (SF), wherein the aliquots (LVA.SVA) comprise a plurality of large-volume aliquots (LVA) and a plurality of small-volume aliquots (SV A), wherein the volume of each of the large-volume aliquots (LVA) is at least 1.5 times and at most 100000 times the volume of each of the small-volume aliquots (SV A),acquiring target molecule presence information, wherein the target molecule presence information is indicative, for each of the aliquots (LVA.SVA), of whether or not the respective aliquot (LVA.SVA) comprises one or more molecules of the target molecule, anddetermining the amount and / or concentration of the target molecule in the sample fluid (SF) based on the acquired target molecule presence information.

14. The method according to claim 13, wherein the volume of each of the large-volume aliquots (LVA) is at least 100 times, at least 150 times, at least 180 times, at least 250 times, at least 350 times, at least 400 times, at least 500 times, at least 750 times, or at least 850 times, or at least 900 times, and / or at most 5000 times, at most 2000 times, at most 1500 times, at most 1200 times, at most 1000 times, at most 800 times, at most 600 times, at most 500 times, or at most 450 times the volume of each of the smallvolume aliquots (SVA).

15. The method according to claim 13 and / or 14, wherein the aliquots comprise a plurality of medium-volume aliquots, wherein the volume of each of the medium-volume aliquots issmaller than the volume of each of the large-volume aliquots and greater than the volume of each of the small-volume aliquots, and wherein, preferably, the volume of each of the large-volume aliquots is at least 1.5 times, at least 2.5 times, at least 3 times, at least 5 times, at least 10 times, or at least 12 times, and / or at most 50 times, at most 30 times, at most 20 times, at most 15 times, at most 10 times, or at most 7 times the volume of each of the medium-volume aliquots, and / or the volume of each of the medium-volume aliquots is at least 10 times, at least 30 times, at least 50 times, at least 100 times, at least 150 times, at least 180 times, at least 200 times, at least 250 times, or at least 280 times, and / or at most 500 times, at most 400 times, at most 350 times, at most 320 times, at most 250 times, at most 230 times, at most 150 times, at most 100 times, or at most 80 times the volume of each of the small-volume aliquots.

16. The method according to one or more of claims 13 to 15, wherein the producing the plurality of aliquots (LVA.SVA) from the sample fluid (SF) comprisesapplying a filling pressure (FP) to the sample fluid (SF) within the at least one microfluidic well (3) of the microfluidic device (1,31) such that the sample fluid (SF) flows into at least a plurality of the large-volume microchambers (10) and into at least a plurality of the small-volume microchambers (11) and, preferably, at least a plurality of the medium-volume microchambers of the at least one microfluidic well (3) as a result of the applied filling pressure (FP), wherein, preferably, the filling pressure (FP) is applied at, at least substantially, the same pressure level or at different pressure levels to the sample fluid (SF) within the at least one microfluidic well (3) when filling the large-volume microchambers (10) and the small-volume microchambers (11) and, preferably, the medium-volume microchambers with the sample fluid (SF) and / or the filling pressure (FP) is applied to the sample fluid (SF) within the at least one microfluidic well (3) by feeding gas via at least one gas flow (GF) into and / or sucking gas via at least one gas flow out of the microfluidic device (1,31) and the at least one microfluidic well (3).

17. The method according to claim 16, comprisingacquiring sample fluid position information, wherein the sample fluid position information represents a position of at least one sample fluid front (SFF) of the sample fluid (SF) within the at least one microfluidic well (3), andcontrolling the filling pressure (FP) applied to the sample fluid (SF) within the at least one microfluidic well (3) based on the sample fluid position information, preferablysuch that the filling pressure (FP) is applied at the different pressure levels to the sample fluid (SF) when filling the large-volume microchambers (10) and the smallvolume microchambers (11) and, preferably, the medium-volume microchambers with the sample fluid (SF).

18. The method according to one or more of claims 13 to 17, wherein the producing the plurality of aliquots (LVA.SVA) from the sample fluid (SF) comprisesfluidly separating the aliquots (LVA.SVA) of the sample fluid (SF) within the microchambers (10,11) of the at least one microfluidic well (3) from each other, preferably by deforming the sealing layer (4) of the microfluidic device (1 ,31) into the channel segments of the at least one microchannel (8,9) fluidly connecting the microchambers (10,11), in particular by applying a compressive force (CF) to the sealing layer (4) and / or after applying the filling pressure (FP) to the sample fluid (SF) within the at least one microfluidic well (3).

19. A system (17,30) comprisingthe microfluidic device (1,31) according to one or more of claims 1 to 12, and a handling apparatus (18,32) configured to handle the microfluidic device (1,31), preferably an analysis apparatus (18) configured to perform the method according to one or more of claims 13 to 18 or a robotic liquid handling apparatus (32).

20. The system according to claim 19, wherein the handling apparatus (18,32) comprises a pressurization (20,33) unit configured to apply a filling pressure (FP) to the at least one sample fluid (SF) within the at least one microfluidic well (3) of the microfluidic device (1,31) such that the sample fluid (SF) flows into at least a plurality of the large-volume microchambers (10) and into at least a plurality of the small-volume microchambers (11) and, preferably, into at least a plurality of the medium-volume microchambers of the at least one microfluidic well (3), and wherein, preferably, the handling apparatus (18,32) comprises an electronic control unit (23,45) configured to control the filling pressure (FP) applied to the at least one sample fluid (SF) within the at least one microfluidic well (3) such that the filling pressure (FP) is applied at, at least substantially, the same pressure level or at different pressure levels to the sample fluid (SF) within the at least one microfluidic well (3) when filling the large-volume microchambers (10) and the small-- 66 -volume microchambers (11) and, preferably, the medium-volume microchambers with the sample fluid (SF).

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