Device for testing biological samples

Abstract

The present invention relates to a fluid network (1) for performing multiple tests of biological samples in parallel, the fluid network having a flow array (2) with a plurality of reaction chambers (14), wherein the reaction chambers (14) respectively have a first channel connector (20) and a second channel connector (21), wherein the first channel connector (20) is connected to a first supply channel (16) and the second channel connector (21) is connected to a second supply channel (19), wherein the first supply channel (16) and the second channel connector (19) are connected to each other by a circulation line (6), wherein at least one component (3, 4, 5, 7) is connected to the circulation line, and a test reagent can be introduced into the reaction chamber (14) of the flow array (2) by means of the component.

Description

Technical Field

[0001] The present invention relates to a device for testing biological samples. Background Art

[0002] Here, a device for testing a biological sample is described, wherein the device is in particular a fluid network for performing a plurality of tests on the biological sample in parallel. The biological sample is in particular a sample together with the nucleic acid sequence contained therein. The test performed by the device is in particular intended for the identification of the nucleic acid sequence or for determining whether a specific nucleic acid sequence is contained in the sample. Such a test is in particular necessary for the decryption of the genetic code (DNA sequencing).

[0003] The deciphering of the genetic code (DNA sequencing) is a standard method in research and medicine.

[0004] A special form of such analysis is so-called next generation sequencing (NGS). The term "next generation sequencing" refers to a whole range of different methods for sequencing, all of which are distinguished primarily by the ability to automatically perform a greater number of tests in a short time (especially also in parallel). For the methods belonging to "next generation sequencing", synthesis sequencing is mainly used.

[0005] In these methods, the DNA molecules to be sequenced are first amplified (clonal amplification) and bound to a solid phase. The identification of a specific nucleic acid sequence is carried out with the aid of a marker (fluorescently labeled and date-labeled nucleotides) which is deposited on the DNA molecule at a position where a specific pattern exists. When the product chain is synthesized, the addition of each fluorescently labeled nucleotide on the DNA is measured, which ultimately allows the sequence to be inferred. The methods performed in these systems are also referred to as synthetic sequencing (SBS).

[0006] In SBS, the sequence is usually read out by periodically executing the following steps:

[0007] 1) Load (= flush + incubate) the reaction area with fluorescently labeled and date-labeled nucleotides.

[0008] 2) Add nucleotides.

[0009] 3) Wash / rinse the reaction area.

[0010] 4) optically detecting the fluorescence event and identifying where the nucleotide was added,

[0011] 5) Load (= flush + incubate) the reaction area with a solution for removing the date-labeled nucleotides.

[0012] 6) Wash / rinse the reaction area.

[0013] The challenge for miniaturization of such sequencing methods is to perform multiple wash and rinse steps in order to deliver a variety of changing fluids to the reaction area. Commercial sequencers, for example, require up to 100 mL [milliliters] of wash solution per sequencing run. Summary of the invention

[0014] Here, a device is described, with which the analysis of biological samples (especially DNA samples) can be efficiently performed after the start of synthesis sequencing. With the described device, in particular, the volume of the various reagents (reagents with nucleotides, washing solutions, etc.) required for sequencing can be significantly reduced.

[0015] The device described here is a fluid network for performing multiple tests of biological samples in parallel, wherein the fluid network has a flow array with multiple reaction chambers, wherein the reaction chambers respectively have a first channel connector and a second channel connector, wherein the first channel connector is connected to a first supply channel and the second channel connector is connected to a second supply channel, wherein the first supply channel and the second channel connector are connected to each other by a circulation pipeline, wherein at least one component is connected to the circulation pipeline, and test reagents can be introduced into the reaction chambers of the flow array by means of the component.

[0016] The core of the described fluid network is the flow array. The flow array contains reaction chambers in which tests can be performed for which the described fluid network is provided. The flow array preferably has a plurality of reaction chambers, for example more than 100 reaction chambers, more than 200 reaction chambers or even more than 1000 reaction chambers.

[0017] The fluid network is particularly related to a microfluidic network. The term "microfluid" here firstly relates to the order of magnitude of a microfluidic device. In this respect, the microfluidic device is particularly characterized in that the physical phenomena usually assigned to microtechnology in the fluid channels and chambers arranged in the microfluidic device are important. This includes, for example, capillary effects, effects related to the surface stress of the fluid (especially mechanical effects). In addition, this also includes effects such as thermophoresis and electrophoresis. These phenomena usually dominate in microfluidics relative to effects such as gravity. The microfluidic device can also be characterized in that it is at least partially manufactured using a layered method and channels are arranged between the layers of the layer structure. The term "microfluid" can also be characterized by a cross section inside the device for guiding the fluid. For example, a cross section in the range of 10 μm [micrometer] × 10 μm to 800 μm × 800 μm is common.

[0018] Preferably, the fluid network is formed in the manner of a Lab-on-Chip. In this respect, the fluid network can, for example, be part of a Lab-on-Chip, for example be arranged in and / or on a chip. Furthermore, the fluid network can be integrated in and / or on a chip.

[0019] The term "channel connector" refers to a connector of the reaction chamber that is connected to a so-called supply channel, which connector serves as a distributor for distributing fluid from a circulation line to the individual reaction chambers of the flow array. In particular, the first channel connector is connected to a common first supply channel and / or the second channel connector is connected to a common second supply channel.

[0020] The circulation line is used for central control of the introduction of reagents into the reaction chamber. Liquid plugs (liquid sections separated by a conveying medium such as oil) in the circulation line can be introduced into the reaction chamber by conveying in the circulation line via supply channels and channel connections.

[0021] The term "component" refers to all components which are used to introduce reagents into the flow array or to control such introduction, for example conveying mechanisms, filters, containers, valves.

[0022] Particularly preferably, the fluid network is designed for testing a biological DNA sample and for identifying nucleic acid sequences in the DNA sample.

[0023] Furthermore, the fluid network is preferred if at least one conveying mechanism is connected to the circulation line, wherein reagents can be conveyed into the reaction chamber via the first supply channel and / or the second supply channel with the conveying mechanism.

[0024] The delivery mechanism is, for example, a piston pump or a diaphragm pump. It is particularly preferred that the delivery mechanism has a delivery direction that can be purposefully reversed, so that delivery in both directions is possible in the circulation line and thus it is possible to influence whether and, if so, when and how a liquid plug with a specific reagent enters a reaction chamber of the flow array.

[0025] It is further preferred that the fluid network is connected to the circulation line if at least one container in which a reagent is provided is connected to the circulation line.

[0026] Particularly preferably, a plurality of such containers are connected to the circulation line. Particularly preferably, on the one hand, there is a container for a reagent, which plays an active role (so-called reaction reagent) in the inspection process that can be performed with the described fluid network. Particularly preferably, there is additionally a container for another fluid, which only has a mechanical or fluid-mechanical effect (such as a conveying medium provided between the liquid plugs) in the operation of the fluid network. This, for example, includes the following fluid, which is substantially inert for the reaction performed within the operating range of the described fluid network. Such a fluid is particularly used to separate reaction reagents from each other. Such a fluid is especially oil.

[0027] The fluid network is particularly preferred if at least one container, in which the displacement medium is provided, is connected to the circulation line.

[0028] Liquids can be specifically expelled with the expulsion medium. In particular, liquids that interact with other reagents and / or samples within the scope of the analytical method performed by the described device can be expelled. Therefore, it is possible to separate reagents from each other and / or separate samples and reagents from each other.

[0029] The displacement medium can be mineral oil, silicone oil, fluorinated oil or derivatives or mixtures of the oils mentioned.

[0030] It is particularly preferred that such containers can be connected to the circulation line in such a way that they can be actively switched on or off in a targeted manner, so that the fluid from the individual containers can enter the circulation line in a targeted manner and thus can also be supplied in a targeted manner to the reaction chambers in the flow array.

[0031] Furthermore, it is preferred if at least one container can be connected to a valve of the circulation line.

[0032] A plurality of valves can also be provided in order to (targetedly) connect the containers. For example, a valve arrangement can be implemented to provide reagents from the respective containers into the circulation line and thus also for processes within the reaction chamber, wherein the circulation line can be switched by the valve arrangement so that it optionally passes by or passes through the respective containers.

[0033] Furthermore, it is preferred if the fluid network has arranged in the circulation line at least one filter which can be used to filter the reagent moving in the circulation line.

[0034] Such a filter can optionally also be connected via a valve of the circulation line. It is used to purify the fluid conveyed in the circulation line.

[0035] Furthermore, the fluid network is advantageous if the reaction chambers in the flow array are arranged in a two-dimensional array (ie, arranged in a two-dimensional array). In other words, this means in particular that the reaction chambers are arranged relative to one another in such a way that they together form a (two-dimensional) matrix.

[0036] Such an arrangement allows a compact and space-saving provision of reaction chambers on the flow array.

[0037] It is further preferred that the fluid network is separated from one another by separating webs in the throughflow array.

[0038] The throughflow array can particularly preferably be embodied as a perforated plate, wherein the individual holes form reaction chambers and the material remaining between the reaction chambers forms separating webs.

[0039] Furthermore, it is preferred if blocking projections are provided in the reaction chamber, with which particles can be retained in the reaction chamber.

[0040] In addition, if a coupling input area for introducing excitation with an excitation unit is arranged on the first side of the flow array and a reading area for optically reading the flow array with a detection unit is arranged on the second side of the flow array, then the fluid network is preferred. The coupling input area is usually set up in this regard for introducing excitation with an excitation unit. The reading area is usually set up in this regard for optically reading the flow array with a detection unit. There are also some optical methods in which the excitation light is generated from the same side and the emitted fluorescence signal is read. When using such a method, the coupling input area and the reading area can also be constructed together on one side (constructed on the first side or the second side of the flow array).

[0041] The first side and the second side (and thus the coupling-in region and the reading region) are arranged on (mutually) opposite, planar sides of the flow array and are preferably located on the side of the first supply channel or the second supply channel facing away from the flow array. The excitation unit is preferably configured (e.g. by radiation) to excite optical markers in reaction chambers of the flow array so that these optical markers emit optical signals. The detection unit (e.g. a camera) is preferably configured to detect such optical signals and thereby also enable evaluation.

[0042] Here, a fluid network is also to be described, in which the flow array is designed as an exchangeable insert. In this respect, the flow array can be formed, for example, in the form of an exchangeable module. In particular, the flow array can be releasably connected to the rest of the described device (the rest of the fluid network). In this respect, it is particularly advantageous if the insert is formed from a material that is different from at least one material of the rest of the device.

[0043] The throughflow array can be designed in particular as a cartridge which can be replaceably introduced into the rest of the described device (described fluid network). By introducing the throughflow array, the reaction chambers of the throughflow array are connected to the supply channels and thus also to the circulation line.

[0044] The fluid network is particularly preferred if the diameter of the reaction chambers in the throughflow array is between 1 nm [nanometer] and 500 μm [micrometer], preferably between 1 μm and 50 μm.

[0045] Reaction chambers of this order of magnitude have proven to be particularly advantageous for the described device (described fluid network) and the experiments that can be performed therewith.

[0046] The core of the described device is a flow array, which can be conceived in particular as a miniaturized well plate. The individual holes of the well plate are reaction chambers in which the individual identification steps can be carried out. Preferably, different nucleotides are arranged in the reaction chambers, on which the DNA molecules to be sequenced are fixed, and the DNA molecules can enter the reaction chambers through supply channels. In addition, the DNA sequences to be sequenced can also be prepared by a process known in the prior art called BEAMing. The beads formed here - on the surface of which multiple DNA chains of the same sequence are bound - can be flushed into the perforated array in the next step and blocked (=fixed) there. By combining a water-insoluble, immiscible phase (e.g. mineral oil) with an aqueous process liquid, advantageous sequencing can be achieved in such a flow array according to the following basic principle: in the first step, the perforations can be loaded / filled with the liquid of the corresponding step, which is carried out through the supply channel. In a next step, the upper and lower sides of the perforated array can be sealed by producing an upper and / or lower layer with oil, which can likewise be done via a supply channel.

[0047] In this fluid state (when the reaction chamber is closed), the corresponding process steps of "sequencing by synthesis" can be performed. Before the next process step of "sequencing by synthesis" is performed, the liquid in the reaction chamber of the previous step can now be directly displaced by means of flushing with oil. As an alternative or in addition, it is possible to work with a water-in-oil plug, whereby a small amount of washing solution (provided and delivered in the form of a plug) can be flushed through the perforations.

[0048] Here, a method for carrying out an analysis of a biological sample is also to be described, the method comprising the following steps:

[0049] a) providing a fluid network according to any one of the preceding claims,

[0050] b) Providing at least one sample in a flow-through array of the fluid network.

[0051] c) Providing at least two different reagents in the fluid network, wherein the two different reagents are separated from one another by a displacement medium.

[0052] d) applying the at least two different reagents to the sample, wherein direct contact of the reagents is prevented by the displacement medium.

[0053] The sample can be introduced into the fluid network in step b), for example together with the flow array. This applies in particular when the sample is implemented in the manner of a cartridge.

[0054] The separation of the different reagents in step c) is preferably carried out in that a liquid plug of a displacement medium is formed in the circulation line of the described device, which liquid plug separates the different reagents from one another.

[0055] The separate loading of the sample with the different reagents in step d) is preferably carried out by displacing the first reagent, preferably completely, from the region of the sample or removing it from the sample by means of the displacement medium before exposing the sample to the second reagent.

[0056] The described fluid network and the described method and the combination consisting in particular of the flow-through array as sequencing area, the use of a water-in-oil mixture and the miniaturization of the array are particularly advantageous. As a result, in particular one or more of the following advantages can be achieved:

[0057] By means of the described device (described fluidic network), all process liquids of a sequencing-by-synthesis method can be reduced. This is a great advantage for sequencing based on a lab-on-a-chip, where only limited amounts of reaction solutions can be pre-stored.

[0058] Not only is the amount of washing reagents required reduced, but also the amount of reaction solutions required, which contain expensive enzymes. This enables dramatic cost reductions.

[0059] The through-flow array allows the excitation and detection units required for the optical readout of the sequencing reaction to be placed on two different sides of the perforated array, which results in improved optical efficiency and more layout possibilities.

[0060] The flow-through array can be designed as an insert (eg a DNA microarray), which means freedom in the choice of materials and advantages in the construction process of one of the flow-through arrays.

[0061] With the described fluidic network, a sample-to-sequencing process is feasible. This has great potential for identifying DNA signatures and even SNPs that cannot be recognized by the probes present on the chip. Since mutations always occur in the target genome, the workflow for sequencing is key to the future reliability of lab-on-a-chip systems.

[0062] With the aid of the described fluidic network, it is possible to allow the sequencing reaction to proceed for such a long time that the gene signature is unambiguously determined. For example, it is also possible to add only a single nucleotide if it is only concerned with the detection of SNPs. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The described device is explained in detail below with the aid of the drawings. The drawings only show a preferred embodiment, and the disclosure of the described device is not limited to the described embodiment. It is shown:

[0064] Figure 1 shows the illustrated fluid network,

[0065] Figure 2 Shown according to Figure 1 The cut section of the fluid network,

[0066] Figure 3 A first embodiment variant of a throughflow array is shown,

[0067] Figure 4 A second embodiment variant of the throughflow array is shown.

[0068] Figure 5 A third embodiment variant of a throughflow array is shown.

[0069] Figure 6 A fourth embodiment variant of a throughflow array is shown, and

[0070] Figure 7The diagram shows a sequence of a method which can be carried out with the aid of the described device. DETAILED DESCRIPTION

[0071] exist Figure 1 , an exemplary fluid network 1 for sequencing is shown in FIG. The fluid network 1 preferably comprises a circulation line 6 and a flow array 2 having a reaction chamber 14. The reaction chamber 14 forms a flow unit, and the fluid circulating in the circulation line 6 flows through the flow unit in parallel. The aggregate of fixed DNA molecules is preferably in the reaction chamber. Sequencing reaction (= decryption of the base sequence) can be performed in each individual reaction chamber 14. The reagent provided for this purpose flows into the first channel connector 20 of the reaction chamber 14 through the first supply channel 16, for example, from the first side 22. Preferably, the first side 22 is the lower side of the flow array 2. That is, the flow array 2 is preferably (geodetically) oriented so that it is passed through from bottom to top. The reagent flows out of the reaction chamber 14 on the second channel connector 21 on the second side 24 of the flow array 2, and then enters the second supply channel 19. Therefore, the first supply channel 16 preferably forms an inflow channel into the reaction chamber 14. The second supply channel 19 thus preferably forms an outflow channel from the reaction chamber 14. The first supply channel 16 and the second supply channel 19 are connected to one another via a circulation line 6.

[0072] The flow array 2 provides a plurality of reaction chambers 14 arranged in parallel to one another. Different containers 5 are connected to the microfluidic network 1 while being separated by valves 3. These containers 5 are prepared with different reagents for sequencing reactions. For synthetic sequencing analysis, for example, labeled nucleotides (e.g. ddNTPs; A=adenine, G=guanine, T=thymine, C=cytosine) are used, which are added to the nucleic acid sequence to be read by a polymerase (e.g. "enzyme 1"). A terminator is fixed to the added nucleotide, which prevents the addition of another nucleotide. In the sequencing reaction, this terminator is removed, for example, by "enzyme 2" before adding another nucleotide. Other reagents are sequencing primers, which define the starting point of sequencing. A washing buffer is present in a further container, with the aid of which the reaction solution in the flow array 2 can be washed away before the next (reaction) step.

[0073] Optionally, the network comprises a filter 7. The components that have been absorbed by the wash buffer can be removed again with the filter 7. The filter 7 can include a dielectrophoretic unit, by which a filtering effect is achieved and with which, for example, nucleotides can be removed from the solution. This enables "wash buffer recovery" to advantageously reduce the provided wash buffer. At least one conveying mechanism 4 is in the network, which can be, for example, a diaphragm pump or a peristaltic pump. The fluid network 1 also has a waste container 11 for receiving reagents that are not reused. The displacement medium is stored in a (not shown) container, which can be used to physically separate the reagents or to directly displace the reagents in the flow array 2 (i.e., a washing aid). The displacement medium can be a derivative or mixed form of mineral oil, silicone oil, fluorinated oil, or the oils mentioned.

[0074] exist Figure 2 An excerpt of an exemplary fluid sequenced in the illustrated fluid network 1 can be seen. Figure 2 The described flow array 2 of the fluid network 1 is shown according to the center of gravity. The reaction chamber 14 described above with the first channel connection 20 and the second channel connection 21 can be seen on the flow array, as well as the first supply channel 16 on the first side 22 of the flow array and the second supply channel 19 on the second side 24 of the flow array. A coupling-in region 25 is arranged on the first side 22, on which the excitation unit 23 can feed the excitation into the reaction chamber 14. Thus, molecules can be excited in the reaction chamber 14 so that they send out optical signals. Such optical signals can be detected by a detection unit 27 on the reading region 26 on the second side 24.

[0075] In the circulation line 6 Figure 2 In the section shown in FIG. 6 , a plurality of phases of the reagents, which are indicated by letters, can be seen. Here, by way of example, aqueous phases (A, D, E, F) can be seen, which are separated from one another by at least one oil phase C (displacement medium). The individual phases (A, D, E, F) form liquid plugs 12 in the circulation line 6, which are separated from one another by phase boundaries 13 and can be specifically conveyed in the circulation line 6 to the flow array 2 or to the reaction chambers 14 contained therein by means of a conveying device (see FIG. 1 ). Figure 1 This is done in that the conveying mechanism carries out the conveying until the corresponding liquid plug 12 is in the reaction chamber 14. To support the use of the conveying mechanism in order to precisely convey the liquid plug 12 into the reaction chamber 14, the actuation unit 23 described further above and the detection unit 27 described further above can also be used.

[0076] A washing buffer (usually an aqueous phase) can also be present before or after the oil phase (or two different phases connected in succession) or in the middle of the two oil phases. DNA aggregates B are respectively fixed on the walls of the cells of the flow array 2 (it will be explained later how and where the DNA aggregates B come from). In this figure, the cells (and the structural space surrounding them) are filled with solution A. In this example, this is, for example, the binding of sequencing primers. The liquid plug C in contact with B is a combination of a washing buffer and a displacement medium. With this washing step, the reagent is removed from B, which is composed of the cells. The multiphase system is now transported for such a long time by the transport unit until the liquid plug D completely fills the cells of the array. In this continuous example, the liquid plug D is a mixture of enzyme 1 and ddNTP "A". If the array is loaded with liquid plug D, the enzyme (polymerase) adds nucleotides for the "T" present in the chain to be read (due to the base pair AT). In the next step, the reaction solution D is washed away with the aid of a wash buffer C after D and the fluorophores present on the added nucleotides are optically detected. This is preferably done with the described excitation unit 23 and the described detection unit 27. Figure 2 The transport mechanism not shown in the figure transports the next liquid-in-liquid plug E into the unit. For example, the liquid plug E is enzyme 2, which removes the terminator of the nucleotide. The washing liquid plug C after E is again a combination of a washing buffer and a displacement medium. The liquid plug F is now transported into the unit with the aid of a pump unit. F is a mixture of enzyme 1 and, for example, ddNTP "C". If the array is loaded with liquid plug F, the enzyme adds nucleotides for the "G" present in the chain to be read (due to base pairing GC). This cycle is performed for a long time until the desired reading length is reached. The exact position of the phase boundary (or in other words, the position of the different liquid plugs) can be detected with the aid of optical control.

[0077] The detection performed by the detection unit is preferably optically controlled. For example, the detection unit is a camera, with which the flow array 2 and / or the circulation line 6 can be monitored, and with which the phase boundaries between the liquid plugs can be identified. The aqueous phase and / or the oil phase can be colored with different dyes or fluorophores (e.g., dyes HEX, FAM; carbon black particles, food dyes, ...) for this purpose. By determining this position, it is possible to control which solutions are reused, which solutions are conveyed to a waste container at the circulation line 6 or to a filter from the circulation line 6. An example of the reuse of a liquid is the enzyme mixture enzyme 1+ddNTP "A", "G", "T", "C". These reagents can be directed back to separate containers so that they can be taken out in a new addition cycle. Similarly, the inert oil plug can be directed back to the corresponding container.

[0078] Three options are thus possible with the described fluid network in order to save buffer volume:

[0079] 1) Reuse the volume by returning it to its container;

[0080] 2) Use two-phase mixtures to reduce dead volume and precisely pump defined volumes;

[0081] 3) Filtration of the washing solution is possible for reusing the washing solution.

[0082] Next, some embodiments are described for providing / immobilizing DNA aggregates for sequencing in aggregates ( Figure 2 , B).

[0083] Figure 3 The cross section of the throughflow array 2 is shown. Individual reaction chambers 14 can be seen, which are separated from each other by separating webs 15 and into which the corresponding reaction liquid can be introduced via first supply channels 16. The reaction chambers 14 form the throughflow cells of the throughflow array. The diameter of the reaction chambers 14 or of the throughflow cells is here between 1 and 500 μm, preferably between 1 μm and 50 μm. DNA primers (“targeted sequencing”) can be pre-stored in the reaction chamber 14 or in the flow cell for generating amplification products in the cell, which are immobilized on the wall of the corresponding reaction chamber 14 / flow cell. Advantageously, another primer is pre-stored in each reaction chamber 14 / flow cell so that different DNA molecules can be read out in different cells. A solution containing at least the DNA molecules to be sequenced and a polymerase is injected into such a flow array 2. This solution is injected into the flow cell / reaction chamber 14 of the flow array 2. For the subsequent reaction, the flow array is sealed with oil “H” so that the first channel connector 20 and the second channel connector 21 (upper and lower sides) of the reaction chamber 14 are closed with oil. This state is also Figure 3 After the amplification reaction (PCR, LAMP, RPA, SDA, ...) is performed, the generated DNA aggregate "G" is fixed on the wall of the corresponding reaction chamber 14. This state is also shown in Figure 3 As described above, the solution in the reaction chamber 14 is sequentially filled and replaced by precisely delivering the fluid in the circulation pipeline.

[0084] Figure 4 Another variant of how the DNA aggregates to be sequenced can be present in the cell is shown. Here, a new form of a flow array 2 with reaction chambers 14 is shown, in which Figure 4Only a detail of the throughflow array 2 is shown. In particular, the first supply channel 16 and the second supply channel 19 are not shown here. Figure 4 The focus is on the illustration of the separate separating webs 15, which separate the individual reaction chambers 14 of the flow array 2 from one another. The reaction chambers 14 are here equipped with blocking projections 17, with which particles 18 can be blocked. Here, the DNA aggregates in the reaction chambers 14 are provided on the particles 18. Within the scope of the BEAMing method, microparticles are generated, on the surface of which the DNA aggregates to be sequenced are immobilized. If by Figure 4 If the solution containing these particles 18 is flushed through the flow array 2 shown, the particles 18 are fixed / positioned by the blocking protrusions 17 present in the flow array 2. Thus, a flow array 2 capable of sequencing is produced by way of example. The diameter of the opening defined by the blocking protrusions 17 is slightly smaller than the diameter of the particles. The diameter of the reaction chamber 14 is here between 1 nm and 100 μm. A flow array containing such particles can be sequenced by means of a multiphase scheme of microfluidics as described above.

[0085] Figure 5 Another possible approach for generating DNA aggregates to be sequenced in the reaction chambers 14 of the flow array 2 is shown. Figure 5 The throughflow array 2 is also only shown very schematically in FIG. Here, at least the inner wall and the edge regions on the upper side and / or the lower side of the cells are coated hydrophilically with a hydrophilic coating 30. The hydrophilic coating 30 on the upper side and the lower side is Figure 5 The remainder of the array is preferably hydrophobic.

[0086] The flow array 2 (as already described) has reaction chambers 14 with a cell diameter of 1 nm to 100 μm. If such an array is loaded with an aqueous phase, the droplets remain in the hydrophilic region, e.g. Figure 5 As shown. This variant can save reaction solution again and increase the cell density additionally. Another advantage is that DNA aggregates can be generated within the scope of digital PCR. The number of DNA molecules to be sequenced is adjusted in this way so that this number is relatively small. If a solution containing at least DNA molecules and polymerase is injected into the cells of the array, the molecules are distributed to the cells according to a Poisson distribution (and then an amplification reaction occurs again, within the scope of which the amplification product is fixed to the wall of the cell).

[0087] Figure 6Another embodiment variant of the flow array is also shown. Here, the cells / reaction chambers 14 of the flow array can be formed with a fabric-like material or a porous material. This has the advantage that the filling of the cells / reaction chambers 14 is supported by capillary forces, the provided reaction volume is further reduced and diffusion washing can be achieved.

[0088] Figure 7 The process of the described method is explained again. Figure 7 2 shows that during the entire sequencing process (or also only in specific steps) one side of the flow array can be closed by an oil phase 28. Thus, the respective aqueous phase 29 of the reagents used for the reaction can be washed through the array on the other side. Here, the washing action is almost exclusively diffusive. This flow principle ("semi-closed array") can also be used for example Figure 4 The flow array shown here further reduces the amount of washing solution required.

[0089] The method can be confirmed by optical inspection of the reaction carrier, in particular the sequencing area, and analysis of the process fluid used.

Claims

1. A fluid network (1) for performing multiple tests of biological samples in parallel, comprising a flow array (2) having a plurality of reaction chambers (14), wherein the reaction chambers (14) each have a first channel connector (20) and a second channel connector (21), wherein the first channel connector (20) is connected to a first supply channel (16) and the second channel connector (21) is connected to a second supply channel (19), wherein the first supply channel (16) and the second supply channel (19) are connected to each other by a circulation line (6), wherein: The reaction chamber (14) forms a flow unit, and the fluid circulating in the circulation line (6) flows through the flow unit in parallel, wherein at least one conveying mechanism (4) is connected to the circulation line (6), and a reagent can be conveyed into the reaction chamber (14) via the first supply channel (16) and / or via the second supply channel (19) by means of the at least one conveying mechanism (4), wherein at least one container (5) is connected to the circulation line (6), and the reagent is provided in the container (5).

2. The fluid network (1) according to claim 1, wherein the fluid network (1) is configured to test biological DNA samples and to identify nucleic acid sequences in the DNA samples.

3. The fluid network (1) according to claim 1, wherein at least one container (5) is connected to the circulation line (6), in which container (5) a pressing medium is provided.

4. The fluid network (1) according to claim 1, wherein at least one container (5) can be connected to the valve (3) of the circulation line (6).

5. The fluid network (1) according to any one of claims 1 to 4, wherein at least one filter (7) is arranged in the circulation line (6), and the reagent moving in the circulation line (6) can be filtered by the filter.

6. The fluid network (1) according to any one of claims 1 to 4, wherein the reaction chambers (14) in the through-flow array (2) are arranged in a two-dimensional matrix.

7. The fluid network (1) according to any one of claims 1 to 4, wherein the reaction chambers (14) in the throughflow array (2) are separated from one another by separating webs (15).

8. The fluid network (1) according to any one of claims 1 to 4, wherein a blocking projection (17) is arranged in the reaction chamber (14), by means of which particles can be retained in the reaction chamber.

9. A fluid network (1) according to any one of claims 1 to 4, wherein a coupling input area (25) for introducing excitation using an excitation unit (23) is arranged on a first side (22) of the flow array (2), and a reading area (26) for optically reading the flow array (2) using a detection unit (27) is arranged on a second side (24) of the flow array (2). 10 . The fluid network ( 1 ) according to claim 1 , wherein the throughflow array ( 2 ) is designed as a replaceable insert.

11. The fluidic network (1) according to any one of claims 1 to 4, wherein the diameter of the reaction chambers (14) in the through-flow array (2) is between 1 nm and 100 μm.

12. A method for performing an analysis on a biological sample, comprising the following steps: a) Providing a fluid network (1) according to any one of claims 1 to 11; b) providing at least one sample in a flow-through array (2) of the fluid network; c) providing at least two different reagents in the fluid network (1), wherein the two different reagents are separated from each other by a displacement medium; d) Loading the sample with at least two different reagents, wherein direct contact of the reagents is prevented by the displacement medium.

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