Receiving unit for receiving fluid, method and apparatus for producing receiving unit, method and apparatus for operating receiving unit, and receiving device

Abstract

To enable reliable filling of a micro-cavity in combination with easy introduction and pre-placement of a reagent dried in the micro-cavity and avoid the risk of carryover of the reagent pre-placed in the micro-cavity during the controlled filling of fluid, e.g., sample liquid into the micro-cavity.SOLUTION: The invention relates to a receiving unit (105) for receiving a fluid, wherein the receiving unit (105) has a receiving element (125) with a receiving face (130) and at least one micro-cavity (135) that is arranged and formed in the receiving element (125) on the receiving face (130) in order to receive the fluid. The receiving face (130) further has a hydrophilic surface characteristic in at least one subregion adjoining the at least one micro-cavity (135).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Background technology The invention relates to a containment unit for containing a fluid, a method and a device for manufacturing the containment unit, a method and a device for operating the containment unit and a containment device according to the independent claims. A computer program is also the subject of the invention.

[0002] For so-called point-of-care molecular diagnostic tests, microfluidic analytical systems, so-called labs-on-a-chip, are particularly suitable, as they allow a fully automated analysis of patient samples. Complex tests often require multiple, mutually independent detection reactions to address different targets in the sample to be examined.

[0003] Summary of the Invention Against this background, the approach presented herein provides an improved storage unit, an improved method according to the main claim, as well as an improved device using this method and finally a corresponding computer program. Advantageous further developments and improvements of the device according to the independent claim are possible by the measures set out in the dependent claims.

[0004] The approach presented here, in contrast to the prior art, allows reliable filling of the microcavities in combination with the simple introduction and pre-deposition of dried reagents in the microcavities, avoiding the risk of carry-over of reagents pre-deposited in the microcavities during the controlled filling of the microcavities with a fluid, e.g., a sample liquid. Similarly, cross-talk of reactions occurring in the fluid-filled microcavities is prevented by sealing the microcavities filled with the sample liquid with a second fluid, i.e., a suitable sealing liquid, for example in order to carry out different, mutually independent amplification reactions in the microcavities, e.g. for the detection of various DNA targets.

[0005] A storage unit for storing a fluid is presented, the storage unit having a storage element with a storage surface having at least one microcavity arranged and formed on the storage surface in the storage element for storing a fluid, and at least one partial region of the storage surface adjacent to the at least one microcavity has hydrophilic surface properties.

[0006] For example, the storage unit can be used in a storage device configured for example for testing a sample. For example, the fluid can be realized as a liquid, for example a sample liquid. The sample liquid can be obtained from biological material, for example of human origin, for example an aqueous solution, for example a body fluid, a smear, a secretion, sputum, a tissue sample or a device with a sample material attached. The sample liquid includes for example medically, clinically, diagnostically or therapeutically relevant species, for example bacteria, viruses, cells, circulating tumor cells, cell-free DNA, proteins or other biomarkers or in particular components of the above-mentioned objects. For example, the sample liquid is a master mix or a component thereof for carrying out at least one amplification reaction, for example in the storage element, for example for DNA detection at molecular level, for example an isothermal amplification reaction or a polymerase chain reaction. The storage element is in particular formed as a sample carrier, the storage surface of which is at least hydrophilic, for example in partial areas adjacent to at least one microcavity. The microcavities arranged in the storage surface can also be referred to as cavities or recesses, for example, and are characterized as cavities with dimensions in the sub-millimeter range. The microcavities may accordingly have hollows so that they can accommodate fluids. Furthermore, the microcavities may be inert and have highly biocompatible surface properties, for example in order to carry out molecular DNA detection reactions therein, such as isothermal amplification reactions or polymerase chain reactions. Capillary and surface forces, for example, are important for the functionality of the device, in particular the overlay of the aqueous phase present in the microcavities with a second immiscible phase. This functionality cannot be guaranteed with large macrocavities.

[0007] According to a particularly advantageous embodiment, the receiving unit has a number of further microcavities that can be arranged and formed in the receiving surface in the receiving element for receiving a fluid. The microcavity and the number of further microcavities can then be arranged in an arrangement area of ​​the receiving surface, in particular in a square, circular, rectangular or elliptical area, in particular at a predefined distance from the edge of the receiving surface, in particular according to a hexagonal, square or rectangular pattern, in which the receiving surface has hydrophilic surface properties, in particular between the microcavity and the number of further microcavities. Due to the predefined distance from the outer border of the arrangement area to the edge of the receiving surface, i.e. the outer edge of the receiving unit, an automatic placement machine (pick-and-place robot) can come into contact with the arrangement area of ​​the microcavities that are particularly relevant for the functionality of the receiving unit, without causing possible contamination of the surface or the microcavities there, and the outer area, the so-called spacing area, can be used during production, for example, for handling the receiving unit by an automatic placement machine (pick-and-place robot). By arranging the microcavities according to a hexagonal pattern, a particularly high surface density of the microcavities can be achieved while maintaining a constant distance between adjacent microcavities. Arranging the cavities in a square or rectangular pattern allows for a particularly simple allocation of the cavities. In an advantageous embodiment, the storage unit further comprises structures adjacent to the storage surface, in particular also outside the area of ​​the arrangement of the cavities, which serve for the allocation or referencing of the microcavities. These are then alignment marks, for example for the standard introduction of reagents into the microcavities using an array spotting system, for example a piezoelectric-based microdispensing system, or for the allocation of the cavities in an optical reader, which detects, for example, a fluorescent signal emanating from a detection reaction in the microcavities of the storage unit. It is further conceivable that different reagents can be introduced or held in different microcavities, for example so that different detection reactions can be carried out in the microcavities.

[0008] According to an embodiment, the microcavity may have at least one sidewall oriented substantially perpendicular to the receiving surface. Advantageously, all sidewalls of the microcavity may also be oriented substantially perpendicular to the receiving surface. This allows, for example, a particularly simple manufacture of the receiving element. The substantially perpendicularly oriented sidewall may, for example, have an angle of 80° to 100° to the receiving surface. Advantageously, this, in combination with a given suitable aspect ratio of the microcavity and / or with additives introduced into the microcavity, allows, upon filling, to reduce the carryover or ejection of, for example, reagents pre-deposited in the microcavity, for example to less than 10% of the amount retained in the microcavity. In particular, for example DNA target-specific primers and / or probes can be pre-deposited in at least one microcavity in which at least one specific detection reaction can be carried out.

[0009] According to an embodiment, the microcavities contain at least one pre-deposited reagent and / or additive. A "reagent" can be understood as a substance used to perform a specific reaction in the microcavity. An "additive", on the other hand, can be understood as a substance generally present in the cavities and allowing the filling of the microcavities and / or reducing the carry-over of the pre-deposited reagent. Thus, the "additive" is particularly decisive for the functionality of the fluid, and the "reagent" is particularly decisive for a correct detection reaction. Advantageously, by pre-depositing in the microcavities in particular an additive, during wetting and filling of the microcavities with the sample liquid, it is possible to avoid undesirable inclusion of air in the microcavities, in particular at the bottom of the microcavities. Furthermore, the at least one pre-deposited reagent can cause a predetermined and desired reaction with the fluid, i.e. in particular the sample liquid, in particular a specific component of the sample liquid, the so-called target, and the sample liquid can be examined for the presence of a specific indicator.

[0010] Particularly advantageously, the storage unit comprises a plurality of microcavities in which at least two different detection reactions can be carried out for detecting at least two different targets. In this way, highly complex molecular diagnostic tests can be carried out in the storage unit, which address a large number of different targets by a large number of different detection reactions. In particular, it is also advantageous in this case to carry out detection in singleplex format in individual fluid partitions of the microcavities, with detection reactions having reduced multiplex performance (geometric multiplexing). Particularly advantageously, mutually independent isothermal DNA detection reactions can be carried out in the microcavities, which on the one hand have a high reaction rate, but on the other hand may only have a low multiplex compatibility (e.g. due to undesirable interactions between primers and / or probes). In this way, a storage unit with a plurality of cavities can be particularly advantageously used in which rapid DNA highly multiplex tests with isothermal detection reactions can be carried out in singleplex format. In particular, multiplex pre-amplification, in particular by polymerase chain reaction, is carried out before the array-based detection in singleplex format in order to increase the sensitivity of the sample analysis. In particular, the detection time for multiplex pre-amplification in a storage unit and singleplex detection of multiple DNA targets is less than 60 minutes in this case, and the detection time for singleplex detection of multiple DNA targets in a storage unit is less than 30 minutes.

[0011] In summary, the storage unit according to the invention allows a very simple and rapid testing of sample liquids for a large number of different targets in an individual device, even with detection reactions, in particular with limited multiplexing capabilities. Advantageously, the use of the storage unit likewise allows for a simple adaptation of multiplex tests, i.e. in particular the addition of detection reactions to multiplex tests, since the detection reactions in the microcavities of the storage unit are carried out independently of one another and accordingly no significant interactions can occur between the different primers and probes used in the microcavities.

[0012] The receiving surface can be at least partially configured as a silicon nitride layer and / or a silicon oxide layer and / or a silane layer, for example a polyethylene glycol-silane layer. Advantageously, the hydrophilicity of the receiving surface allows or significantly improves the penetration of fluids into at least one microcavity, and such a type of receiving surface can be produced by a technically simple, low-cost and mature method. Furthermore, the improvement of the penetration of fluids into the microcavities, especially in combination with the pre-positioning of at least one reagent, especially an additive, in the microcavities and / or the hydrophilic coating of the microcavities, can allow the receiving element to be used in combination with a microfluidic device to allow a fully automated introduction of fluids into at least one microcavity of the receiving unit.

[0013] According to further embodiments, the receiving element and / or the receiving unit can also be constructed from a silicon substrate. The silicon substrate can be realized, for example, as a silicon wafer. For example, such substrates are already used in semiconductor technology and manufacturing methods of semiconductor technology can be used for the manufacture of the approach presented here, which can, for example, reduce the material costs during production. In particular, by processing the silicon wafer, a number of receiving units can be produced in parallel. Furthermore, within the scope of the method for producing the receiving unit described below, a predefined break point can be processed in the silicon substrate simultaneously with the etching of the microcavities. In this way, a particularly simple and low-cost separation of the silicon substrate into a number of receiving units and thus a low-cost production of the receiving units is possible by mechanical destruction of the silicon substrate. Furthermore, due to the high thermal conductivity of silicon, a particularly uniform and rapid temperature control of the microcavities can be achieved in particular by using silicon as substrate material for the receiving units. In this way, a high comparability and rapid execution of the individual detection reactions is provided.

[0014] According to an embodiment, the receiving unit may comprise a further number of microcavities for receiving the fluid, which may be arranged and formed in the receiving surface in the receiving element, in a further arrangement area of ​​the receiving surface, in particular in a square, circular, rectangular or elliptical area, in particular at a distance from the edge of the receiving surface, in particular according to a hexagonal, square or rectangular pattern. Between the arrangement area and the further arrangement area, interval areas without microcavities may be arranged. Here too, a group of microcavities can be used for carrying out multiplex tests, for example when preparing individual microcavities in which different reagents are held. Advantageously, this allows, for example, multiple tests to be carried out simultaneously, for example by pre-arranging further reagents in the further microcavities.

[0015] According to an embodiment, the spacing area can have a width that can correspond, for example, to at least twice the minimum distance between adjacent microcavities in the placement area or the further placement area. Advantageously, this allows the placement areas to be clearly and recognizably differentiated from one another, facilitating the evaluation of the individual microcavities. Furthermore, the spacing area facilitates the handling of the chips after separation of the receiving units.

[0016] According to one embodiment, the microcavities or groups of microcavities have different dimensions and / or different volumes. Due to the different volumes of sample liquid in the individual microcavities, i.e. reaction compartments, there are statistically different numbers of target units, e.g. DNA copies, in compartments of different sizes. Accordingly, in order to achieve reliable detection of different targets in the sample liquid using specific detection reactions with different detection properties, smaller reaction compartments are used for detection reactions with high sensitivity, while larger reaction compartments are used for detection reactions with low sensitivity. In addition, in this way, a larger quantification range can be achieved by using digital detection methods.

[0017] The receiving surface may according to an embodiment have optically recognizable indicia which may have a predetermined position relative to the arrangement of the at least one microcavity, in particular the optically recognizable indicia may have predetermined properties with respect to their size and / or optical properties, advantageously allowing for example to mark reference points and therefore improving automatic readability.

[0018] Further, a storage device is presented, comprising a storage unit of one of the variants presented above, a housing for accommodating the storage unit, a chamber for introducing at least one fluid, e.g. a sample liquid, into at least one microcavity of the storage unit and optionally subsequently introducing a second fluid, i.e. a sealing fluid, which is immiscible or only slightly miscible with the sample liquid and enables covering / sealing of the sample liquid enclosed in the microcavity of the storage device, and at least one passage configured to supply the sample liquid to the microcavity of the storage unit and subsequently cover the microcavity with the sealing fluid and / or enable evacuation and / or drain excess sample liquid and sealing fluid.

[0019] The housing can be formed, for example, to protect the storage unit and the sample liquid from environmental influences and / or conversely to prevent contamination of the environment by the sample liquid. The passage can be realized, for example, in the form of a tube or hose and can, for example, have an approximately rectangular cross section. The storage device can be manufactured, for example, from polymeric materials, for example polycarbonate (PC), polypropylene (PP), polyethylene (PE), cycloolefin copolymers (COP, COC), polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), or thermoplastic elastomers (TPE), such as polyurethane (TPU) or styrene block copolymers (TPS), or combinations of polymeric materials, at low cost and by high-throughput techniques, such as, for example, injection molding, thermoforming, punching, and / or using joining techniques, such as, for example, laser transmission welding.

[0020] Optionally, the receiving device may comprise a pumping device, which may be configured to pump at least one fluid, for example the sample liquid and / or the sealing fluid, through the passage. Advantageously, this may allow a fully automated microfluidic processing. The pumping device may be driven, for example by the processing unit, via a pneumatic interface. The pumping device is in particular based on an elastic membrane, which is integrated in the receiving device and which can be deflected by applying a positive or negative pressure to a recess in the receiving device, thereby achieving a controlled expulsion of the sample or the sealing fluid. In this way, microfluidic elements such as pump chambers and valves can be realized. By appropriately sequentially activating the elements of the pumping device, a controlled transport of the sample liquid and the sealing fluid can be achieved, in particular by means of a peristaltic pump mechanism. Furthermore, the receiving device in particular comprises at least one opening for inputting the sample and optionally a further opening serving as a vent. In an advantageous embodiment, the receiving device comprises further recesses for pre-positioning liquid or solid reagents and a microfluidic network serving the processing of the reagents in the receiving device.

[0021] Furthermore, a method for manufacturing a containment unit in one of the variants presented above is presented, the method comprising a preparing step and a processing step, in which the preparing step comprises preparing a containment surface of the containment element, and in the processing step at least one microcavity is processed in the containment surface for containing a fluid in order to manufacture the containment unit.

[0022] Alternatively or additionally, a photosensitive resist layer / photoresist may be applied in a sub-step of the processing step and / or a lithography sub-step may be provided, structuring may be performed by means of deep reactive ion etching (Bosch process) for processing microcavities (and / or further optically detectable features). The photosensitive resist may be, for example, spin-coated and exposed in a lithography step, after which excess material may be removed. For example, after processing at least one microcavity, the receiving element may be treated, for example so that excess photosensitive resist may be removed. Alternatively or additionally, a coating of the receiving surface and / or the microcavities may also be performed in any step to form hydrophilic surface properties of the receiving surface and / or the microcavities.

[0023] Thus, the method can optionally modify the surface properties of the receiving surface and / or the microcavities to be hydrophilic, for example by forming a silicon nitride or silicon oxide surface and / or a silane layer, for example a polyethylene glycol-silane layer. Alternatively or additionally, it is particularly preferred if in a further optional step a reagent is introduced into the microcavities of the receiving unit. Optionally, the method can also include a splitting step, which can for example split the receiving element. Splitting can be achieved in particular by machining a predetermined breaking point into the receiving surface of the receiving element, which is advantageously carried out together with machining of the microcavities, followed by mechanical destruction.

[0024] Furthermore, a method for operating a storage unit in one of the aforementioned variants is presented, which comprises the steps of filling and sealing, performing and evaluating. In the filling and sealing steps, at least one microcavity is first filled with a sample liquid and then overlaid with a sealing fluid as a second fluid, so that a partition of the sample liquid is present in at least one microcavity as a fluid reaction compartment. The sealing fluid is, for example, a liquid with low water solubility to prevent undesired mixing with the sample liquid, and / or high flowability, i.e. low viscosity to achieve good removal of bubbles formed, for example during thermal treatment of the device, and / or low conductivity to keep parasitic heat losses occurring during thermal temperature control as low as possible, and / or low heat capacity to keep the thermal mass processed, for example during the performance of a polymerase chain reaction, as low as possible, and / or which contains a surfactant to stabilize the interface with the sample liquid. The sealing fluid is, for example, a fluorinated hydrocarbon.

[0025] In the performing step, at least one reaction is performed in the at least one microcavity to obtain a reaction result. To perform the at least one reaction, the reaction compartment present in the storage element, in particular in the at least one microcavity, has in particular a predefined temperature that allows the reaction, for example an isothermal amplification reaction, to proceed. Optionally, in the performing step, the storage device is thermally cycled, for example to perform a polymerase chain reaction in the at least one reaction compartment. In particular, in the performing step, a fluorescent signal emitted from the at least one reaction compartment is also recorded, from which conclusions can be drawn about the progress of the reaction.

[0026] In the evaluation step, the reaction result is evaluated, in particular on the basis of the fluorescent signals recorded in the execution step, and advantageously, the evaluation of the reaction result is performed on the basis of the fluorescent signal profile already in parallel with the execution of at least one reaction, and the execution of the reaction is stopped once the reaction result can be determined with sufficient accuracy.

[0027] Particularly preferred are embodiments of the methods presented herein, in which mutually independent, in particular different, detection reactions are carried out in the microcavities.Alternatively or additionally, a step of multiplex pre-amplification of the sample material and subsequent detection of targets in a singleplex array format can be carried out in the variants of the storage units presented herein.

[0028] Variations of the methods presented herein may be implemented, for example, in software and / or hardware, or in a mixed form of software and hardware, for example in a controller.

[0029] Moreover, the approach presented herein creates an apparatus configured to execute, control or implement the steps of the method variations presented herein in a corresponding device, which also allows the embodiment of the invention in the form of an apparatus to quickly and efficiently solve the problem underlying the invention.

[0030] For this purpose, the device may comprise at least one calculation unit for processing signals or data, at least one storage unit for storing signals or data, at least one interface to sensors or actuators for reading sensor signals from sensors or outputting data or control signals to actuators, and / or at least one communication interface for reading or outputting data embedded in a communication protocol. The calculation unit may be, for example, a signal processor, a microcontroller, etc., and the storage unit may be a flash memory, an EEPROM, or a magnetic storage unit. The communication interface may be configured to read or output data wirelessly and / or wired, and a communication interface capable of reading or outputting wired data may read or output this data from or to a corresponding data transmission line, for example electrically or optically.

[0031] The device in this case can be understood as an electrical device that processes the sensor signal and outputs a control signal and / or a data signal accordingly. The device can have an interface that can be configured on a hardware and / or software basis. In the case of a hardware-based configuration, the interface can be part of a so-called system ASIC, which has various functions of the device. However, it is also possible for the interface to be an integrated circuit in itself or to consist at least partially of separate components. In the case of a software-based configuration, the interface can be a software module that is present, for example, on a microcontroller alongside other software modules.

[0032] In an advantageous design, the device provides control of the method for operating the storage unit. For this purpose, the device has access to sensor signals, e.g. read signals representative of the read information and / or control signals for controlling any step of the method. The control is provided by actuators, e.g. a reading unit, an evaluation unit and / or a providing unit.

[0033] Also advantageous is a computer program product or computer program having a program code which can be stored on a machine-readable carrier or storage medium, such as a semiconductor memory, a hard disk memory or an optical memory, and which is used for executing, implementing and / or controlling the steps of the method according to one of the above-mentioned embodiments, in particular when the program product or program is run on a computer or device.

[0034] Examples of the approach presented herein are illustrated in the drawings and explained in more detail in the following description. [Brief description of the drawings]

[0035] [Figure 1] 1 is a schematic side view of a containment device according to one embodiment; [Figure 2A] 1 is a schematic side view of a containment unit according to one embodiment; [Figure 2B] FIG. 2 is a schematic top view of a containment unit according to one embodiment. [Diagram 3] 1 is a schematic side view of a containment unit according to one embodiment; [Figure 4] 1A-1D are schematic diagrams of different stages of an intermediate product of a possible manufacturing process of a containment unit according to one embodiment; [Diagram 5] 1 is a flow diagram of a method for manufacturing a containment unit according to one embodiment. [Figure 6A] FIG. 2 is a perspective view of a storage unit according to one embodiment. [Figure 6B] FIG. 13 is a perspective view of a storage unit according to a further embodiment; [Figure 7] FIG. 2 is a diagram illustrating a procedure for determining the reaction result of a polymerase chain reaction obtained in a containment unit according to one embodiment. [Figure 8] FIG. 13 shows reaction results obtained in a containment unit according to one embodiment after carryover testing. [Figure 9] FIG. 1 is a diagram illustrating a procedure for determining the reaction results of a multiplex test obtained in a containment unit according to one embodiment. [Figure 10] 1 is a flow diagram of a method of operating a storage unit according to one embodiment. [Figure 11] FIG. 2 is a block diagram of an apparatus according to one embodiment.

[0036] In the following description of preferred embodiments of the invention, elements shown in the various figures and having similar functions will use the same or similar reference numerals, and repeated descriptions of these elements will be omitted.

[0037] 1 shows a schematic side view of a containing device 100 according to an embodiment. The containing device 100 is configured to introduce a fluid into the containing unit 105 and / or to cover the containing unit 105 of the containing surface 130 at least in its partial areas, in particular in the arrangement area of ​​the cavities, with a further fluid, a so-called sealing fluid, in particular after introducing the fluid into the containing unit 105. For this purpose, the containing device 100 comprises a containing unit 105 for containing a fluid, a housing 110 for containing the containing unit 105, a chamber 115 configured for introducing the fluid into the containing unit 105, and at least one passage 120 configured for supplying the fluid to the containing unit 105 and / or for draining the fluid from the containing unit 105 and / or for allowing evacuation of the chamber 115 and the microcavities 135, 150. Optionally, the containing device 100 comprises a pump device configured for pumping the fluid and possibly the sealing fluid through the at least one passage 120. The storage unit 105 comprises a storage element 125 having a storage surface 130 formed with hydrophilic surface properties, and at least one microcavity 135 arranged and formed in the storage surface 130 of the storage element 125 for storing a fluid.

[0038] According to the present embodiment, the receiving element 125 is, for example, composed of a silicon substrate. The receiving surface 130 is, for example, at least partially composed of a silicon nitride layer, a silicon oxide layer and / or a silane layer, for example a polyethylene glycol-silane layer, for example to facilitate the penetration of the fluid into the microcavities 135. The microcavities 135 have, according to the present embodiment, side walls 140 oriented substantially perpendicularly to the receiving surface 130, for example oriented at an angle 145 of 80° to 100° to the receiving surface 130. According to an alternative embodiment, the microcavities 135 are formed approximately cylindrical. Furthermore, optionally, the receiving unit 105 comprises, according to the present embodiment, a number of further microcavities 150 arranged and formed in the receiving surface 130 in the receiving element 125 for receiving the fluid. In so doing, the microcavity 135 and the plurality of further microcavities 150 are arranged in an arrangement area not shown here, in particular in a square, circular, rectangular or elliptical area of ​​the receiving surface, in particular at a predefined distance from the edge of the receiving surface, in particular according to a hexagonal, square or rectangular pattern, so that the receiving surface 130 has hydrophilic surface properties, in particular between the microcavity 135 and the plurality of further microcavities 150. Furthermore, optionally, the receiving unit 105 according to this embodiment has an optically recognizable feature 155 having a defined position relative to the arrangement of the at least one microcavity 130. That is to say, the optically recognizable feature 155 according to this embodiment has predefined properties with regard to size and optical properties.

[0039] In other words, a microcavity array chip, i.e. a storage unit 105, is presented for filling the microcavities 135, 150 in the storage unit 105 with sample liquid by dispensing a fluid, also called sample liquid, i.e. wetting the storage surface 130 with the sample liquid and successively wetting the storage surface 130 with a sealing fluid, such that the sample liquid remains at least partially in the microcavities 135, 150 of the storage unit, and for carrying out mutually independent reactions in partitions of the sample liquid present in the microcavities 135, 150 after dispensing of aliquots, i.e. sample liquid, each of which may contain specific reagents pre-deposited in the microcavities 135, 150. Thus, the approach presented herein relates to a device for distributing a sample liquid in a number of compartments, also called microcavities 135, 150, as well as the carrying out of a number of mutually independent reactions in the microcavities 135, 150. In particular, the distribution of fluids and the performance of reactions are performed automatically, for example in a microfluidic system, referred to as a containment device 100 according to this embodiment.

[0040] Thus, the approach described herein produces a solution that, according to this embodiment, allows for example dry reagents to be easily introduced and pre-positioned in the microcavities 135, 150 by the containment unit 105, has sufficiently low carryover of pre-positioned reagents during controlled dispensing of fluids into the microcavities 135, 150, has negligible reaction cross-talk between different microcavities 135, 150, enables (automatable) microfluidic dispensing of fluids into the microcavities 135, 150, and can be manufactured at low cost and / or can be integrated into the containment device 100 such that fully automated microfluidic processing is achieved.

[0041] The containment device 100 in particular comprises, according to the present embodiment, a chamber 115 with advantageous predetermined dimensions 160, provided for introducing a fluid into the microcavities 135, 150 and / or sealing the microcavities 135, 150 with a second fluid immiscible with the fluid. The microfluidic chamber 115 comprises, according to the present embodiment, at least one passage 120, also referred to as supply and / or discharge passage, provided for the controlled supply or discharge of a fluid to the containment unit 105. In an advantageous design of the containment device 100, the containment device 100 further comprises a passage system not shown here and / or a pump device not shown, in order to allow a fully automated microfluidic processing of the containment unit 105.

[0042] In this case, the receiving unit 105 has a receiving surface 130, which according to the present embodiment is also called a plane and which has an arrangement of microcavities 135, 150 processed in a receiving element 125 formed from a substrate material. According to the present embodiment, the receiving surface 130 then has a hydrophilic wettability, in particular in the immediate vicinity of the microcavities 135, 150. The microcavities 135, 150 according to the present embodiment are characterized in particular by substantially vertical side walls 140, in particular the receiving surface 130 is associated with an angle 145 of approximately 90° to the side walls 140 of the microcavities 135, 150 at the microcavities 135, 150 or at their openings. The microcavities 135, 150 optionally in particular contain at least one pre-deposited substance, also called a reagent or additive. The microcavities 135, 150 optionally have an approximately cylindrical shape, which allows the receiving unit 105 to be particularly simply manufactured. The arrangement of the microcavities 135, 150 in particular follows a square, hexagonal or rectangular pattern, in order to optionally enable a standard introduction of reagents into the microcavities, in particular with an array spotting system, in particular with a piezoelectric-based microdispensing system. Simply optionally, the receiving surface 130 according to this embodiment additionally has optically recognizable features 155, for example with a defined position relative to the arrangement of the microcavities (20) and with suitable properties in terms of size and optical properties. Thus, the features 155 are detectable in particular by an optically sensitive device, such as a camera of the array spotting system, and are applicable for a defined and fully automated introduction of reagents into the arrangement of the microcavities 135, 150. Alternatively or additionally, the features 155 are applicable for determining the relative positions of the microcavities 135, 150, in particular when performing an optical evaluation of the reactions taking place in the microcavities 135, 150.

[0043] In summary, the approach presented herein describes a containment unit 105 in combination with a hydrophilic containment surface 130 with which the fluid comes into contact at least in a partial area for filling the microcavities 135, 150, at least partially vertical side walls 140 of the microcavities 135, 150, in particular against carry-over of reagents pre-deposited in the microcavities 135, 150, pre-deposited reagents enabling the performance of different specific detection reactions in the microcavities 135, 150, and / or at least one pre-deposited additive, such as a substance that wets the microcavities 135, 150, ensures complete filling so that air is not trapped in the microcavities 135, 150, and / or results in a reduction in carry-over of the above-mentioned reagents pre-deposited in the microcavities 135, 150, as well as the use of cavities 135, 150 with a solid bottom. This means that there are no perforations, making it easier to pre-position the reagent and / or at least one additive in the cavities 135,150.

[0044] The approach presented here, in addition to the microfluidic functionality with regard to filling and / or sealing of the reaction compartments, according to this embodiment, ensures low crosstalk of the reactions taking place in the compartments, i.e. microcavities 135, 150. Furthermore, the approach presented here accounts for the wettability of the microcavities 135 (containing, for example, polyethylene glycol as a dry additive, containing primers and probes for molecular DNA detection reactions as dry reagents and / or having a silicon oxide layer, a silicon nitride layer or a silane layer as hydrophilic surface) and the containing surface 130, consisting for example of silicon nitride, silicon oxide or a hydrophilic silane layer, in particular a polyethylene glycol-silane layer, of the flow cells, consisting for example of a polymer, for example of polycarbonate.

[0045] Alternatively, components manufactured, for example, in an alternative manner not described herein, may likewise be used to provide the functionality referred to herein, but in this case the storage unit 105 will be somewhat more complex to manufacture than a storage unit 105 manufactured according to the approach described herein, for example involving two lithography steps.

[0046] In order to prevent or reduce fluid crosstalk between adjacent compartments, the prior art in particular uses devices with hydrophobic surface properties between the compartments. However, this has the disadvantage that the hydrophobic top surface makes it difficult to fill the compartments in the substrate. In particular, the prior art uses cavities with sloping side walls or perforations or low aspect ratio morphologies to allow easy filling of the compartments with the aqueous phase when the top surface is hydrophobic and the size of the compartments is small, for example when the lateral dimensions / diameters of the compartments are in the sub-millimeter range. However, cavities with sloping side walls have a relatively small volume in relation to their surface area. This is disadvantageous for the execution of highly multiplexed amplification reactions, especially when the reactions are evaluated optically, since on the one hand it is desirable to have as high a surface density of reactions carried out in parallel as possible, and on the other hand, since the small volume of the compartments results in a relatively weak fluorescent signal, which reduces the signal-to-noise ratio during optical evaluation. Also, cavities with sloping side walls are difficult to pre-deposit reagents in, since the flow profile created when the compartment is filled with sample liquid tends to cause unwanted carryover of pre-deposited reagents. Again, perforations have the disadvantage that they only allow deposition of reagents on the side walls of the perforations, making it difficult to introduce and pre-deposit reagents into the individual reaction compartments.

[0047] 2A shows a schematic side view of a storage unit 105 according to an embodiment. The storage unit 105 shown here may correspond or be similar to the storage unit 105 described in FIG. 1. According to this embodiment, the storage unit 105 is shown simply in an enlarged view in which at least one pre-deposited reagent 200 according to this embodiment is visible in the microcavity 135. That is, the storage unit 105 according to this embodiment contains at least one pre-deposited reagent. Furthermore, according to this embodiment, it is clearly shown that the centers of the microcavities 135, 150 have a distance 205 with respect to the adjacent microcavities 135, 150 that is the same as the distance between the center of the optically recognizable element 155 and the center of each adjacent microcavity 135, 150.

[0048] In other words, a containment unit 105 is described that allows for distributing fluids into and performing multiple independent reactions within the microcavities 135, 150, in which the dry reagents 200 are pre-disposed. Additionally, a method for manufacturing the containment unit 105 is presented, which is illustrated in one of the following figures. In particular, the containment unit 105 allows for reliable introduction of reagents 200 into the microcavities 135, 150, significantly reduces carryover of reagents 200 pre-deposited in the microcavities 135, 150, e.g. to <10%, when dispensing fluids into the microcavities 135, 150, the containment unit 105 has very low (<1%) cross-talk of reactions between different microcavities 135, 150 after sealing of the microcavities with an appropriate sealing fluid, allows automatable microfluidic dispensing of fluids within the microcavities 135, 150, and can be incorporated into microfluidic systems such as the containment device 100.

[0049] Thus, the containment unit 105 has, according to the present embodiment, microcavities 135, 150 which serve for the construction of microfluidic compartments. The microcavities 135, 150 in this case have, in particular at the interface with the side of the containment unit 105 in contact with the fluid, a pre-deposited reagent 200 and a limited aspect ratio, in particular to prevent undesired air inclusions in the microcavities 135, 150 when filling the microcavities 135, 150 with a fluid and to allow complete filling of the microcavities 135, 150 with a fluid. The containment surface of the containment unit 105 which comes into contact with the fluid and through which the filling of the microcavities 135, 150 takes place, has, according to the present embodiment, hydrophilic surface properties, in particular in the immediate vicinity of the microcavities 135, 150, in order to allow the penetration of the fluid into the microcavities 135, 150. The containment unit 105 can particularly advantageously be part of a containment apparatus as illustrated in Fig. 1 in order to allow fully automated microfluidic processing and possibly carrying out reactions in the microcavities 135, 150. According to this embodiment, this allows for reliable filling due to the hydrophilic surface nature of the containment surfaces of the containment unit 105 adjacent to the microcavities 135, 150, pre-deposition of reagents 200 and / or additives in the microcavities 135, 150, as well as suitable aspect ratios of the properly obtained fluid-containing microcavities 135, 150.

[0050] Furthermore, the (almost) vertical side walls of the microcavities 135, 150, e.g. in combination with suitable additives, can minimize the carryover of pre-deposited reagents 200 during filling with a fluid, e.g. to <10%. This is especially true in comparison with microcavities 135, 150 with sloping side walls, the geometry of which, in relation to the resulting flow profile, in principle leads to a larger carryover of the reagents 200 pre-deposited in the microcavities 135, 150. Furthermore, in the approach presented herein, e.g. by sealing the microcavities 135, 150 after e.g. filling the microcavities 135, 150 with a suitable second fluid that is immiscible with the fluid, it is possible to achieve a small crosstalk between adjacent reaction compartments, e.g. <1%, when carrying out chemical reactions in the microcavities 135, 150. This allows for spatially separated reactions to be carried out in the microcavities 135, 150, independent of each other. The resulting geometric multiplexing allows the fluid to be tested for a large number of different targets, for example if suitable target-specific detection reagents are pre-deposited in the microcavities 135, 150. Moreover, according to this embodiment, the containment device particularly advantageously allows for fully automated microfluidic processing. In particular, the containment device used for processing the containment unit 105 can be manufactured at low cost from a polymer or a combination of polymer materials. In this way, the functionality provided by the containment unit 105 is realized in a miniaturized lab-on-a-chip system that can be used for molecular laboratory diagnostics.

[0051] 2B shows a schematic top view of a receiving unit 105 according to an embodiment. The receiving unit 105 comprises micro-cavities 135, 150 arranged according to a hexagonal pattern in a circular placement area 600. Furthermore, the outer boundary of the placement area 600 of the micro-cavities 135, 150 (shown by a dashed dotted line) has a predefined minimum distance to the edge of the receiving surface of the receiving unit 105. This edge area can in particular be used to enable an easy handling of the receiving unit 105 by an automatic placement machine (pick-and-place robot), thereby enabling for example an easy manufacturability of the receiving device 100 described above. Furthermore, the storage unit 105 has in this embodiment an optically recognizable feature 155, also referred to as a reference mark, which can be used, for example, for unambiguous allocation and / or symbolic designation of the microcavities 135, 150 and / or for positioning the storage unit 105 in a processing device, for example with an optical detection system, for example in a microdispensing system for automated introduction of reagents into the microcavities 135, 150, and / or for positioning in a processing device that can detect, for example by means of an optical system, in particular a fluorescent signal, for example a fluorescent signal profile of a biochemical reaction in the microcavities 135, 150.

[0052] Fig. 3 shows a schematic side view of a storage unit 105 according to one embodiment. The storage unit 105 shown here may correspond or be similar to the storage unit 105 described in Fig. 1 or Fig. 2. The only difference is that the close-up view according to this embodiment does not show optically visible elements.

[0053] Figure 4 shows a schematic diagram of different intermediate product stages of a possible manufacturing process 400 of a storage unit 105 according to an embodiment. The storage unit 105 may in this case correspond or be similar to the storage unit 105 described in figures 1 to 3 and is therefore also applicable to a storage device as described in figure 1.

[0054] In this case, according to the present embodiment, a receiving element 125 made of silicon, also called silicon wafer, is used as substrate material. First, a hydrophilic surface property of the substrate material is formed on the receiving surface 130. According to the present embodiment, this is in particular a silicon nitride surface, which can be produced on a silicon substrate, for example, by means of a method of depositing silicon oxide, silicon nitride and polysilicon, also called low pressure chemical vapour deposition (LPCVD), as well as metals. In particular, according to the present embodiment, a layer system made of, for example, 50 nm SiO2 and 140 nm Si3N4 is suitable for producing a low-distortion Si3N4 layer with good adhesion to the silicon substrate. According to the present embodiment, silicon nitride is suitable as a surface coating, since it has particularly hydrophilic wetting properties. For example, after a pretreatment with hexamethyldisilazane (HMDS), a photosensitive resist (Fotolack) 405, also called photoresist (Photoresist), is applied, which serves as a mask for etching microcavities into the silicon substrate. According to the present embodiment, after the exposure 410 of the photosensitive resist 405 to define the structures to be etched, the resist is developed. Subsequently, according to the present embodiment, the Si3N4 and SiO2 are removed in the exposed areas 420, for example by means of a CF4 dry etching 415. The microcavities 135, 150 are processed in the silicon substrate, for example by means of a deep reactive ion etching 425. Advantageously, the deep reactive ion etching 425 is process-technically optimized for the production of microstructures with approximately vertical sidewalls. The remaining photosensitive resist 405 is removed, for example by treatment in an oxygen plasma 430. The introduction of one or more reagents 200 into the microcavities 135 is according to the present embodiment carried out, for example, by means of a piezoelectric-based microdispensing system or an array spotting system. Particularly advantageously, such a manufacturing process 400 can be carried out at wafer level, allowing a particularly low-cost parallel production of the containing units 105.Separation of the parallel manufactured storage units 105 can be carried out, in particular after introduction of the reagents 200 into the microcavities 135, for example by sawing, breaking or using another, for example laser-based, separation method, for example so-called Mahoh-Dicing.

[0055] Figure 5 shows a flow diagram of a method 500 for manufacturing a containment unit according to one embodiment. The illustrated method 500 includes eight sub-steps 502 according to the manufacturing process 400 illustrated in Figure 4, and can manufacture a containment unit as illustrated in any of Figures 1 to 3. According to this embodiment, the method 500 includes a step 505 of preparing a containment surface and a step 510 of machining at least one microcavity in the containment surface for containing a fluid, in order to manufacture the containment unit.

[0056] According to one embodiment, steps 505, 510 and / or sub-step 502 of method 500 may be omitted and / or performed in a different order in advantageous embodiments.

[0057] FIG. 6A shows a perspective view of a semi-finished product in the manufacture of a containing unit 105 according to an embodiment. The containing unit 105 shown here may correspond or be similar to the containing unit 105 described in any of FIGS. 1 to 3. According to this embodiment, a plurality of further microcavities 150 are formed for containing a fluid. Here, according to this embodiment, the microcavity 135 and the plurality of further microcavities 150 are arranged in a substantially square arrangement area 600 according to a square pattern. In this case, the containing surface 130 has hydrophilic surface properties, in particular between the microcavity 135 and the plurality of further microcavities 150.

[0058] According to this embodiment, it is further evident that the containing unit 105 comprises, in addition to the microcavity 135 and the plurality of further microcavities 150, a plurality of further microcavities 605 formed for containing a fluid. According to this embodiment, the plurality of further microcavities 605 are arranged in the further arrangement area 610 so as to form a square, rectangular or hexagonal shape, in particular between the arrangement area 600 and the further arrangement area 610 a spacing area 615 is arranged in which no microcavities 135, 150, 605 are provided. According to this embodiment, the spacing area 615 has a width which corresponds for example to at least twice the minimum distance between adjacent microcavities of the arrangement area 600 or the further arrangement area 610.

[0059] In other words, according to this embodiment, a diagram of a processed silicon wafer having micro-cavities 135, 150, 605 is shown after performing a method for fabricating a containment unit 105, for example as described in FIG.

[0060] 6B shows a perspective view of a silicon substrate with predefined break points machined to form a number of storage units before separating the substrate. The storage units each comprise a microcavity and a number of further microcavities arranged according to a hexagonal pattern in a (nearly) circular arrangement area. Furthermore, the storage units each have optically recognizable indicia for the introduction of a reagent into the microcavity, for example by means of a microdispensing system, and / or for the positioning of the storage unit in a detection device, and / or for the unambiguous designation of the microcavities.

[0061] 7 shows a diagram for explaining a procedure for determining a reaction result 700 of a polymerase chain reaction obtained in a storage unit 105 according to one embodiment. Such a reaction result 700 can be obtained in a storage unit 105 as described in any one of the above-mentioned Figs. 1 to 3.

[0062] In other words, for example, as sample liquid, also referred to as fluid, a so-called PCR master mix was used, containing the target gene at a concentration of 10 initial copies per microcavity (25 nl), which further contained a target-specific TaqMan fluorescent probe (Cy3) indicating the amplification of the target gene.

[0063] 7a shows a schematic fluorescence micrograph of the fluids dispensed into the microcavities of the containment unit 105, which may also be referred to as a device, taken during temperature cycling for performing a polymerase chain reaction. Microcavities containing fluids in which a significant amount of PCR product has already been generated appear bright, e.g. due to cleavage of the fluorescent probe. Microcavities that do not contain a significant amount of PCR product appear dark according to this example.

[0064] FIG. 7b shows a signal profile specific to microcavity "F3" with a sigmoidal increase due to the polymerase chain reaction occurring within this microcavity.

[0065] Figure 7c shows the normalized sigmoidal fitted amplification curves of the individual microcavities summarized in a graph. According to this example, 89 out of 96 microcavities show an increase in the fluorescent signal, with an average ci value, i.e. PCR cycle at the inflection point of the sigmoidal signal increase, of 31.53, with a standard deviation of 0.81 temperature cycles. Four microcavities according to this example do not show a significant increase in the fluorescent signal during 50 temperature cycles. The remaining three microcavities have an increase in the fluorescent signal with a ci value >45 temperature cycles. This is shown by a histogram of the ci values ​​in Figure 7d.

[0066] Figure 7e illustrates this by a map showing the spatial distribution of ci values ​​in an appropriate pseudocolor representation. According to Figures 7c, 7d and 7e, it is clear that amplification occurs in the majority of microcavities (92.71%) in the ci value range of 30-34 temperature cycles. The variation in the measured ci values ​​can be partly due to the statistical variation of the copy number initially present in the microcavities. Based on the binomial distribution, for an average initial copy number of 10 copies per microcavity, a variation of about 2-16 initial copies per microcavity can be estimated, which corresponds approximately to the four PCR cycles mentioned above. On the other hand, the number of negative cavities is not only due to the statistical variation of the copy number in the cavity based on the binomial distribution. Here, the amplification properties of the detection reaction, especially the sensitivity, the lower limit of detection, play a decisive role. Microcavities with a negative signal profile are due to the fact that amplification does not necessarily occur when a low number of copies is initially present in the microcavities. The sensitivity of the selected detection reaction is too low for this. In additional measurements, the statistical detection limit of the reactions used here was determined to be about 2.5 initial copies per microcavity, the so-called lower detection limit. Moreover, microcavities with a negative signal profile show, according to this example, that no significant copy numbers are found in them that are generated by PCR, even after the amplification reactions have proceeded in the neighboring microcavities. As a result, these microcavities can be used as indicators of crosstalk between neighboring reaction compartments. In particular, the three microcavities in which delayed PCR occurs may be relevant. That is, the delay in the sigmoidal increase for more than 10 PCR cycles is not due to an initial statistical fluctuation in the copy number, according to this example. Rather, in this case, these may be delayed positive or false positive amplification reactions that may have been initiated by crosstalk of amplification reactions in neighboring microcavities.Based on the fact that the array has microcavities with a negative signal profile and that an increase in false positive reactions occurs only with a delay of 10 PCR cycles, according to this embodiment, the crosstalk between reactions performed in adjacent microcavities is 100% per amplification cycle.

number

[0067] 8 shows a schematic diagram of a reaction result 800 obtained in a storage unit 105 according to one embodiment after carryover testing. Such a reaction result 800 can be obtained in a storage unit 105 as described in any of the previously presented Figures 1 to 3.

[0068] According to this embodiment, carryover of reagents pre-deposited in the microcavities is checked, which may occur during the so-called microfluidic processing of the containing unit 105, i.e. the controlled filling of the microcavities with a fluid and the subsequent controlled sealing of the microcavities with a second fluid. For this purpose, according to this embodiment, copies of a target gene, e.g. the ABL gene, are introduced in (approximately) every other microcavity in a checkerboard pattern using a microdispensing system / array spotting system, pre-deposited in dry form, e.g. with polyethylene glycol (PEG) as additive (see FIG. 8a).

[0069] Figure 8b shows a schematic of fluorescence micrographs taken during temperature cycling. The images were taken after some microcavities already showed a significant increase in the fluorescence signal, indicating the generation of PCR products. A stronger fluorescence signal is observed in microcavities pre-deposited with about 100 copies of template DNA of the target gene (patterned filling in Figure 8a) than in microcavities without pre-deposited template DNA (unfilled in Figure 8a). This therefore indicates selective amplification and thus a slight carry-over of pre-deposited reagents during microfluidic processing.

[0070] FIG. 8c shows an additional spatial distribution of ci values ​​according to this embodiment. In the microcavities each pre-disposed with 100 copies of template DNA, robust amplification is observed with ci values ​​of 26.8-28.8 temperature cycles. Meanwhile, in the remaining microcavities, no amplification can be observed in most cases within 50 temperature cycles. Delayed amplification with a delay of more than 4 temperature cycles occurs only in 8 microcavities. As a result, the carryover of reagents pre-disposed in the microcavities of the containing unit 105 during microfluidic processing of the containing unit 105 is reduced by up to about ½. 4 = 1 / 16 = 6.25%. Therefore, the containing unit 105 is also suitable for carrying out multiplex amplification reactions in which target-specific reagents, such as primers and probes, are pre-positioned in the microcavities.

[0071] FIG. 9 shows a diagram illustrating a procedure for determining a reaction result 900 of a multiplex test obtained in a storage unit 105 according to an embodiment. Such a reaction result 900 can be obtained in a storage unit 105 as described in any of the previously presented FIGS. 1 to 3. According to this embodiment, a reaction result 900 obtained from a multiplex test using pre-arranged primers and probes is shown. For this purpose, according to this embodiment, target-specific primers and probes corresponding to the two target genes "ABL" and "e13a2" were pre-arranged in dry form, for example with polyethylene glycol as an additive, in each of the 12 microcavities of the storage unit 105. The probes had fluorophores corresponding to "Cy3" and "Cy5" as shown diagrammatically in FIG. 9a. A PCR master mix with a concentration of 100 copies of ABL template DNA per microcavity, for example 25 nl, was introduced as sample liquid and the process was performed.

[0072] Figure 9b, 9c show schematic representations of two fluorescence images taken before and after thermal cycling. The figures shown consist of two individual fluorescence micrographs taken with filter sets corresponding to the fluorophore Cy3, shown in the horizontal pattern, and the fluorophore Cy5, shown in the vertical pattern, respectively. No significant carryover of reagents pre-deposited in the microcavities, or cross-talk of reactions occurring in adjacent microcavities, is visible in the images. Only the microcavities pre-deposited with primers and probes have clear fluorescence signals. Thus, according to this example, the images confirm previous experiments described in Figure 8 regarding low carryover during microfluidic processing and negligible cross-talk between adjacent microcavities of the device presented here.

[0073] FIG. 9d shows the sigmoidal signal profile of microcavity “G4” indicating positive detection of ABL template DNA in the sample liquid by polymerase chain reaction.

[0074] Figure 9e shows a map of the spatial distribution of ci values. In exactly 12 microcavities where primers and probes for the ABL target gene were pre-positioned, amplification can be observed with ci values ​​ranging from 27.3 to 29.6.

[0075] Figure 9f shows a graph corresponding to the normalized amplification curves of 12 microcavities. Taken together, the measurements highlight the extremely high suitability of the containment unit 105 for carrying out geometrically highly multiplexed sample analyses using molecular diagnostic amplification methods.

[0076] Fig. 10 shows a flow diagram of a method 1000 of operating a containment unit according to one embodiment. The method 1000 can be used for a containment device as described in Fig. 1. The method 1000 in this case comprises a step 1005 of filling and sealing at least one microcavity with a fluid or with a second (sealing) fluid, which is for example immiscible or only slightly miscible with the fluid, a step 1010 of carrying out at least one possible reaction in the at least one microcavity to obtain a reaction result, and a step 1015 of evaluating the reaction result.

[0077] In other words, the filling of the microcavities of the receiving unit with a fluid is performed. The microcavities already filled earlier with the fluid are then sealed with a second (sealing) fluid, which is immiscible or only slightly miscible with the fluid. In particular, the second fluid, also called sealing fluid, is a fluorinated hydrocarbon. Furthermore, according to this embodiment, mutually independent reactions, in particular amplification reactions, for example polymerase chain reactions or isothermal amplification reactions, are carried out, for example in order to detect at least one target gene in the microcavities of the receiving unit. If necessary, suitable reaction conditions for this are established by external influences, for example heat input or heat release. In a particularly preferred embodiment, steps 1005, 1010, 1015 are carried out automatically in a processing unit provided for the processing of the receiving device.

[0078] According to one embodiment, steps of method 1000 may be omitted and / or performed in a different order in advantageous embodiments.

[0079] Fig. 11 shows a block diagram of an apparatus 1100 according to an embodiment. According to this embodiment, the apparatus 1100 is configured to execute or control any of the methods described in Fig. 5 or Fig. 10. The apparatus 1100 is for example configured to read an input signal 1105 using a reading unit 1110 and to provide a control signal 1115 using a providing unit 1120. According to this embodiment, the apparatus optionally comprises an evaluation unit 1125 configured to evaluate information represented by the read signal 1105.

[0080] Where an example includes an "and / or" or "and / or" connection between a first feature and a second feature, this should be read as meaning that the example according to one embodiment has both the first feature and the second feature, and that an example according to a further embodiment includes only the first feature or only the second feature.

[0081] An example specification is shown below: Thickness of the receiving element (125): 100 μm to 3000 μm, preferably 300 μm to 1000 μm Lateral dimensions of the receiving element (125) or receiving surface (130): 3mm x 3mm to 30mm x 30mm, preferably 5mm x 5mm to 15mm x 15mm Number of microcavities (135) and further microcavities (150): 2 to 2000, preferably 50 to 500 Microcavity (135) capacity: 1nl to 100nl, preferably 5nl to 40nl Diameter of microcavity (135): 100 μm to 500 μm, preferably 250 μm to 400 μm Depth of microcavity (135): 100 μm to 500 μm, preferably 200 μm to 300 μm Aspect ratio (depth to diameter) of the microcavity (135): 0.3 to 1.0, preferably 0.6 to 0.7 Distance between an edge of a microcavity (135) and an edge of at least one further microcavity (150) adjacent to the microcavity (135): 70 μm to 300 μm, preferably 100 μm to 200 μm Contact angle of water on the receiving surface (130): <10°~75°, preferably <10°~40° Reagents pre-disposed in the microcavities (135, 150): target-specific primers and probes, template DNA; Additive: Polyethylene glycol (PEG) with a molecular weight of, for example, 6000 or 2000, and a concentration in solution of 2-5% (w / v) Fluid (sample liquid): A master mix for an amplification reaction such as PCR or isothermal amplification, or a master mix that does not contain its components, in particular the primers and / or probes present in the microcavities (135, 150). Second Fluid (Sealing Fluid): Fluorinated hydrocarbons such as 3M Fluorinert FC-40, Fluorinert FC-70, or Novec 7500 Flow rates for filling and sealing the microcavities (135, 150) of the containment unit (105) in the containment device (100) for microcavities (135, 150) of 350 μm diameter and 240 μm depth, with the chamber (115) having suitable dimensions such as 7 mm×7 mm×1 mm (volume approximately 50 μl): 5~10μl / s

Claims

1. A storage unit (105) for storing a sample liquid mainly composed of polar molecules, A containment element (125) comprising a containment surface (130) and at least one microcavity (135) disposed and formed on the containment surface (130) of the containment element (125) for containing the sample liquid, The receiving surface (130) has a hydrophilic surface property in at least one partial region adjacent to the at least one microcavity (135), To accommodate the sample liquid, the containment element (125) comprises a plurality of further microcavities (150) arranged and formed on the containment surface (130), wherein the containment surface (130) has hydrophilic surface properties between the microcavity (135) and the plurality of further microcavities (150). The microcavity (135) has a side wall (140) oriented substantially perpendicular to the housing surface (130), The microcavity (135) comprises at least one pre-configured additive and at least one pre-configured reagent (200), wherein the additive is configured to avoid the undesirable sealing of air into the microcavity (135) and to reduce carryover from the pre-configured reagent (200). The substantially vertically oriented side wall (140) is at an angle of 80° to 100° with respect to the housing surface (130), and water forms a contact angle of up to 75° in the portion having the hydrophilic surface properties adjacent to at least one of the microcavities (135). The aforementioned additive is particularly PEG (polyethylene glycol), and is pre-placed in a dry form together with the pre-placed reagent (200). The microcavity (135) and the plurality of further microcavities (150) are each sealed with a sealing fluid that is immiscible with the sample liquid after the sample liquid has been introduced into the microcavity (135) and the plurality of further microcavities (150). Storage unit (105).

2. The housing unit (105) according to claim 1, wherein the housing surface (130) is at least partially composed of a silicon nitride layer, a silicon oxide layer, or a silane layer.

3. The housing unit (105) according to claim 1 or 2, wherein the housing element (125) is made of a silicon substrate.

4. The housing unit (105) according to any one of claims 1 to 3, wherein the microcavities (135) and the plurality of further microcavities (150) are arranged in a square, rectangular, round, elliptical, circular or hexagonal shape in the arrangement area (600).

5. The housing unit (105) according to any one of claims 1 to 4, wherein the housing surface (130) has an optically recognizable mark (155) having a predetermined position relative to the arrangement of the at least one microcavity (135), and in particular the optically recognizable mark (155) has predetermined properties with respect to its size, shape and / or optical properties.

6. The housing unit (105) according to claim 5, wherein the optically recognizable mark (155) includes a reference mark.

7. The housing unit (105) according to any one of claims 1 to 6, wherein the microcavities (135) and the plurality of further microcavities (150) are arranged in a square, rectangular, round, elliptical, circular or hexagonal shape in a placement area (600), the partial region having the hydrophilic surface properties exists only between the microcavities (135) and the plurality of further microcavities (150), and a spacing area (615) without microcavities is formed between the placement area (600) and the further placement area (610).

8. The containment unit (105) according to any one of claims 1 to 7, wherein the sealing fluid is a fluorinated hydrocarbon.

9. A storage device (100), A storage unit (105) according to any one of claims 1 to 8, A housing (110) for housing the aforementioned housing unit (105), A chamber (115) configured to introduce the sample liquid into the containment unit (105), At least one passage (120) configured to supply the sample liquid to the containment unit (105) and / or discharge it from the containment unit (105), A housing device (100) equipped with the following.

10. A method (500) for manufacturing a housing unit (105) according to any one of claims 1 to 8, Step (505) of preparing the housing surface (130) of the housing element (125), To manufacture the containment unit (105), the process includes a step (510) of machining a microcavity (135) for containing the sample liquid into the containment surface (130), A method (500) for manufacturing a housing unit (105), including the above.

11. A method (500) for manufacturing a housing unit (105) according to claim 10, wherein a deep reactive ion etching method is used in the processing step (510).

12. A method (1000) for operating a housing unit (105) according to any one of claims 1 to 8, The steps include introducing a sample liquid into at least one microcavity (135), and then sealing the microcavity (135) with a sealing fluid (1005), To obtain the reaction results (700; 800; 900), the step (1010) of carrying out at least one reaction in the microcavity (135), Step (1015) to evaluate the reaction results (700; 800; 900), A method (1000) for operating a housing unit (105), including the above.

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