DE202018006869U1 - Microfluidic system for digital polymerase chain reaction (dPCR) of a biological sample - Google Patents

Abstract

Microfluidic system (1; 1') for the digital polymerase chain reaction dPCR of a biological sample, the microfluidic system (1; 1') comprising:
at least one microfluidic device (2) with an inlet (23), an outlet (24), a flow channel (25) which is arranged between the inlet (23) and the outlet (24) and the inlet (23) with the outlet (24) connects, and an arrangement of reaction areas (26) which are in fluid communication with the flow channel (25);
a flow circuit (3) connectable to the microfluidic device (2) to allow liquid to flow through the flow channel (25) of the microfluidic device (2);
a sample liquid source which can be connected to the microfluidic device (2) in order to supply the flow channel (25) and thus the arrangement of reaction regions (26) of the microfluidic device (2) with a sample liquid (27) via the inlet (23);
a primary separation liquid source connectable to the microfluidic device (2) to supply the flow channel of the microfluidic device (2) via the inlet (23) with initial separation liquid (28) for sealing the sample liquid (27) within the array of reaction regions (26 ) to supply;
a secondary separation liquid source (4) which can be connected to the microfluidic device (2) in order to supply the flow channel (25) of the microfluidic device (2) with additional separation liquid (29) via the inlet (23),
a pump device (31) which is connected to the flow circuit (3) and is suitable for pumping the additional separating liquid (29) through the flow channel (25), and
a control device in the form of a programmable logic controller for executing a computer-readable program with instructions for carrying out operations, which is set up to control the pumping device (31) in order to pump the additional separating liquid (29) through the flow channel (25) if necessary, to flush gas bubbles from the flow channel (25).

Description

TECHNICAL FIELD

The present invention relates generally to the technical field of sample analysis, such as the study of chemical or biochemical reactions, and more particularly to the technical field of high-throughput analysis of biological samples. More specifically, the present invention relates to a microfluidic system for digital polymerase chain reaction (dPCR) of a biological sample. Specifically, such a system includes a microfluidic device with a flow channel that is in fluid communication with an arrangement of reaction areas, which are often also referred to as subdivisions and are implemented as reaction chambers or reaction vessels, for example in the form of depressions or microwells, which serve as reaction sites for chemical or biological reactions of at least one biological sample provided therein, and wherein the system can achieve a desired thermocycling temperature profile in the microfluidic device as quickly and as reliably as possible.

BACKGROUND

In the field of diagnostic technology for the study of chemical or biochemical reactions, a goal is to be able to carry out several different examinations on one or more test samples on the same - preferably disposable microfluidic device, thereby enabling methods for the independent analysis of one or more test samples with several different reagents in the course of a single analysis process. As such test samples, biological samples are typically used, which are often collected by medical personnel from patients for laboratory analysis, e.g. to determine the concentrations of various components in the collected samples. Accordingly, the terms "sample" and "biological sample" refer to material(s) that may contain an analyte of interest, which sample may be from any biological source, such as a physiological fluid, including blood, Saliva, eye lens fluid, cerebrospinal fluid, sweat, urine, stool, sperm, milk, ascitic fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cultured cells or the like, the sample being particularly suspected of containing a specific antigen or nucleic acid.

Most of the known chemical, biochemical and/or biological assays involve immobilizing a biological sample, as mentioned above, within reaction sites and performing one or more reactions on the immobilized sample material, followed by a quantitative and/or qualitative analytical procedure. For many biological, biochemical, diagnostic or therapeutic applications, it is essential to be able to accurately determine the amount or concentration of a particular substance or compound in the biological sample, i.e. the analyte of interest. In order to achieve this goal as accurately as possible, various methods have been developed over the years in this technical field, such as the well-known polymerase chain reaction (PCR), which allows the in vitro synthesis of nucleic acids in a biological sample, whereby a DNA segment can be specifically amplified, i.e. a cost-effective way to copy or amplify small DNA or RNA segments in the sample. The development of such methods for reproducing DNA or RNA sections has brought enormous advantages in genetic analysis as well as in the diagnosis of many genetic diseases or in the detection of viral load.

Typically, thermal cycling, also known as thermocycling, is used to heat and cool the reactants in the sample within the reaction chamber to amplify such DNA or RNA segments, with laboratory equipment including thermal cyclers commonly used to achieve an automatic To achieve a method for diagnostic assays based on PCR, in which during a PCR procedure the liquid PCR samples must be repeatedly heated and cooled to different temperature levels and must be kept at different temperature plateaus for a certain time. In a typical PCR method, for example, a particular target nucleic acid is amplified through a series of repetitions of a cycle of steps in which the nucleic acids (a) present in the reaction mixture are denatured at relatively high temperatures, for example at a denaturation temperature of more than 90 ° C , typically about 94°C to 95°C, to separate the double-stranded DNA, then b) the reaction mixture is cooled to a temperature at which short oligonucleotide primers bind to the single-stranded target nucleic acid, for example at a hybridization temperature of about 52° C to 56 °C for primer binding to the separated DNA strands to provide templates (annealing or hybridization), and then (c) the primers are extended using a polymerase enzyme, for example at an extension temperature of about 72 °C Formation of new DNA strands so that the original nucleic acid sequence is replicated. Repeated cycles of denaturation, hybridization and elongation, typically about 25 to 30 repeated cycles, result in an exponential increase in the amount of target nucleic acid present in the sample. In order to be able to precisely maintain such temperature plateaus during thermocycling, a uniform temperature distribution should be maintained across the reaction zone so that all reaction areas can be heated and cooled uniformly in order to obtain a uniform sample yield between the reaction areas containing the sample. To carry out a regular PCR method, well-known thermocycling devices, such as thermocyclers / thermal cyclers, can be used to amplify DNA sections, which essentially consist of a holder for holding the samples, often also referred to as a sample temperature control holder, and a heat pump attached to the holder, the heat pump often being in the form of a combination of a Peltier element, which is used to actively heat and cool the holder and thus to actively control the temperature supplied to the samples, and a corresponding heat sink, which is connected to the Peltier element. Element is thermally coupled in order to dissipate the heat and, for example, conduct it into the environment.

In general, there is a need to carry out diagnostic tests ever faster, more cost-effectively and more easily and to achieve a high level of precision while simultaneously increasing the efficiency of conventional laboratory processes. A particular example of the mentioned methods for the amplification of DNA or RNA segments, which are the focus of the present invention, is the digital polymerase chain reaction method, also referred to as digital PCR or dPCR, which is a biotechnological further development of the conventional PCR method described above. Method and can be used for the direct quantification and cloning amplification of nucleic acids, including DNA, cDNA or RNA. The main difference between dPCR and conventional PCR essentially lies in the method for measuring the amounts of nucleic acid, since conventional PCR carries out one reaction per individual sample, while dPCR carries out a single reaction within a sample that is divided into a large number of partitions , which are provided in a correspondingly large number of reaction areas, the reaction being carried out individually in each reaction area, so that a more reliable detection and more sensitive measurement of the nucleic acid amounts is possible. Accordingly, significant efforts have been made to achieve miniaturization and integration of various assay procedures to increase the number of parallel assays on a single support device. As an example of such a single carrier device, microfluidic devices, such as microfluidic chips, have been developed which have microchannels and microreaction areas which accept samples on a microliter or nanoliter scale in the form of flowable sample liquid, such as aqueous sample liquid. Microliter-scale reagents, typically prefilled into an array of small wells, i.e., microwells or nanowells, provided as reaction areas on the microfluidic chip, are placed therein to flow with a stream of sample liquid flowing through a flow channel Each type of assay depends on the reagents loaded into the array of reaction areas as well as the configuration of flow channels and detectors, wherein the filling of sample liquid into the microfluidic chip can be done by pipetting the sample liquid into the chip . This advanced technology allows a variety of tests to be performed simultaneously on a miniaturized scale. Most of these chemical, biochemical and/or biological assays aim to immobilize biological materials such as polypeptides and nucleic acids, cells or tissues in the wells and perform one or more reactions on the immobilized material, followed by a quantitative and/or qualitative analysis procedure , such as luminescence test measurements. Shows for illustrative purposes 3A a top view of an example of a known microfluidic chip 6 in a schematic manner, and 3B shows chip 6 of 3A in a cross-sectional view along a line AA in 3A . The microfluidic chip 6 consists of a lower plate 61 and an upper plate 62, ie a so-called two-layer microfluidic chip, where the upper plate 62 has an inlet 63 for the introduction of liquid into the chip and an outlet 64 for the exit of liquid from the Chip provides and the combination of lower plate 61, ie the lower layer of the chip, and upper plate 62, ie the upper layer of the chip, forms therebetween a flow channel 65 which connects the inlet 63 to the outlet 64, wherein an array of micro-wells 66 is provided in the flow channel 65 on its upper side, ie on the inside of the upper plate 62. Here, when filling a sample liquid through the inlet 63 into the flow channel 65 towards the outlet 63, the sample liquid is filled into each element of the array of microwells 66 as the sample liquid flows along the array of microwells.

1. (a) beim Befüllen der mikrofluidischen Vorrichtung mit Probenflüssigkeit und Trennflüssigkeit können Gasblasen in der mikrofluidischen Vorrichtung eingeschlossen werden;
2. (b) während des Thermocyclings können anfänglich eingeschlossene Gasblasen (siehe (a)) aufgrund der Verdampfung der Probenflüssigkeit wachsen; oder
3. (c) durch die Verdampfung der Probe können neue Gasblasen entstehen.

However, when the reaction chamber volumes are actually miniaturized into microfluidic structures of a microfluidic device to produce the desired small dimensions, several already known problems increase, such as problems related to the increased surface area to volume ratio or the undesirable evaporation of sample liquid and in particular the undesirable generation of gas bubbles in the liquid provided in or flowing through the microfluidic device. Gas bubbles in a liquid within a microfluidic structure can pose a serious problem because gas bubbles circulating through a microfluidic system can -as a side issue- not only damage the microfluidic structure of any type of sensor used within it, but more importantly can harm the biological sample of interest by causing undesirable mixing of samples in adjacent microwells, leading to cross-contamination and therefore significant experimental errors and false test results. For example, gas bubbles can lead to experimental errors on chromatography columns by causing the reaction components within the reaction areas to dry out. In addition, gas bubbles can severely impair the visual recognition of the reaction areas and the reactions taking place therein, which can lead to failed assays. In particular, when performing dPCR of samples in the microfluidic device, gas bubbles such as air bubbles tend to occur frequently due to various reasons as follows:

1. (a) when filling the microfluidic device with sample liquid and separation liquid, gas bubbles can be trapped in the microfluidic device;
2. (b) during thermocycling, initially trapped gas bubbles (see (a)) may grow due to evaporation of the sample liquid; or
3. (c) new gas bubbles can form due to the evaporation of the sample.

To illustrate the above-mentioned gas bubble formation and gas bubble growth is shown in the 4A until 4D a microfluidic chip 7 is shown schematically in cross section, the chip 7 being inserted into a corresponding thermocycling device, also shown only schematically. The microfluidic chip 7 has a similar structure to the microfluidic chip 6 3A & 3B , ie with a lower plate 71 and an upper plate 72, and with an inlet 73, an outlet 74 and a flow channel 75 connecting the inlet 73 to the outlet 74, in the flow channel 75 at its top, ie on the inside the upper plate 72, an arrangement of reaction areas 76 in the form of micro-wells is provided. The arrangement of the reaction areas 76 is already filled with sample liquid 77 and a separating liquid 78 has been introduced into the flow channel 75, which seals the sample liquid 77 within the reaction areas and fills the flow channel 75 from the inlet 73 to the outlet 74. In order to be able to carry out thermocycling of the microfluidic chip 7, the chip 7 is arranged in the thermocycling device in the form of a so-called plate cycler 8, ie a thermocycler or thermal cycler with a lower heater 81 and an upper heater 82, which are heated by heatable plates, too Called heating plates, are realized. In 4A the microfluidic chip 7 is shown in an unheated state of the heating elements 81, 82, whereby when filling the microfluidic device 7 with sample liquid 77 and/or separating liquid 78, a gas bubble 91, for example an air bubble or the like, is accidentally released into the flow channel 75 immediately below the arrangement of the reaction areas 76 was introduced.

Accordingly, "initial" gas bubbles 91 can be trapped inside the chip 7 during filling, for example in the sample liquid located in a microwell or in a microwell, or in sealing liquid or separating liquid located in the flow channel of the chip 7. As in 4B can be seen, in which at least the floor heater 81 was switched on in order to achieve a heating temperature of >50 ° C, the already introduced gas bubble 91 grows due to the evaporation of the sample liquid 77 within the arrangement or array of reaction areas 76. In addition, a new gas bubble 92 due to the undesirable evaporation of the sample liquid 77 within the arrangement of the reaction areas 76. Here the new gas bubble 92 can arise and grow during the temperature control in each of the reaction areas and emerge from it into the flow channel 75. In 4C a progressive growth of the gas bubbles 91, 92 can be seen due to undesirable evaporation of the sample liquid 77, with the flow channel 75 showing bubble growth along its extent, so that the growing gas bubbles 91, 92 gradually remove the separating liquid 78 from the flow channel 75 and thus from the Press out inlet 73 and outlet 74, shown here by corresponding arrows. As a result, the growing gas bubbles 91, 92 force the separating liquid 78 out of the microwells, which means that the sample liquid 77 can evaporate more easily in the microwells. The growing gas bubbles 91, 92 can also extend over more than one micro-well, ie Bubbles 91, 92 can push the separating liquid 78 away from several micro-wells and thus “expose” these micro-wells as a whole, so that the contents of one micro-well can pass into an adjacent micro-well. As the heating by the thermal cycler continues, the bubbles 91, 92 can continue to grow and also fuse to form a large gas bubble 93, see 4D , which leads to the fused and growing gas bubble 93 gradually pushing the separating liquid 78 further out of the flow channel 75 and thus further out of the inlet 73 and the outlet 74, which in turn is shown by corresponding arrows. Through the in the 4A until 4D shown development 4A to 4D, especially in 4D , it can be clearly seen that the separating liquid 78 is almost completely pushed away from the arrangement of the reaction areas 76 by undesirable gas bubble formation and growth, as a result of which the separating effect of the separating liquid 78 is canceled. Accordingly, the reaction components in the sample liquid 77, which is located within the arrangement of the reaction regions 76, are no longer preserved by the separating liquid 78 and can therefore dry out. In other words: 4A until 4D show that during thermocycling, “initial” gas bubbles 91 and newly formed gas bubbles 92 can grow due to the evaporation of the sample liquid 77 provided within the reaction areas 76, with the gas bubble growth continuing until the flow channel 75 and/or the in the form of the arrangement of Reaction areas 76 provided measuring area is largely emptied. Accordingly, cross-contamination between adjacent reaction areas can occur due to the removal of the separating liquid 78 by the gas bubbles 91, 92, 93. In addition, the gas bubbles 91, 92, 93 can increase the shear stress on the biological sample material within the reaction regions 76 as the cell membranes stretch under the force of the undesirable liquid-air interface thus created. Furthermore, as previously mentioned, the gas bubbles can pose a significant problem for the visual detection of the reaction areas and the reactions taking place therein, essentially resulting in a failed assay test that must be avoided at all costs. Therefore, eliminating or avoiding gas bubbles is a major challenge in the present technical field.

To date, there have been various approaches to solving the problem of gas bubbles in the flow channel of a microfluidic device. Like for example in EP 2 830 769 A1 described, one solution to the gas bubble problem is to prevent gas bubbles from entering the microfluidic device during filling by providing certain microfluidic structures after an inlet, such as providing a slot running along an entire edge of a substrate is designed to allow liquids to flow from an inlet manifold through the slot, substantially around the entire edge of the substrate, and into a reaction chamber at the same pressure and without gas bubbles. However, a significant disadvantage of such a solution is that if such microfluidic structures do not prevent gas bubbles from entering the microfluidic device, the gas bubble can end up in the reaction chamber and grow due to the evaporation of the sample at elevated cycle temperature without any possibility consists of removing the gas bubble that has entered. Additionally, a new gas bubble may be created by evaporation of the sample at elevated cycling temperatures, causing the dPCR method to fail. Accordingly, even if the known solution can prevent existing gas bubbles from penetrating into the microfluidic device more or less effectively, gas bubbles that still enter or newly generated gas bubbles cannot be handled during thermocycling.

Another solution to the problem of gas bubbles is, for example, from US 2005/0009101 A1 known, in which microfluidic cassettes or devices are described with which a series of manipulations can be carried out on a sample, which ultimately lead to the detection or quantification of the target analyte, with a light guide being provided which enables the detection of substances formed in hybridization liquid or washing buffer bubbles, and wherein a roller is provided in functional relationship with a flexible layer for peristaltic interaction to remove any detected bubbles, using an elastomeric material of the flexible layer as a peristaltic material. Accordingly, the proposed solution is aimed at a way of squeezing bubbles of any kind from the microfluidic device, which, however, requires an elastomeric fluidic chip material. However, such a solution is not considered suitable for a dPCR chip because the handling and filling of such a flexible chip material is ineffective and inaccurate, and the surface modifiability and optical quality of such a material are poor compared to the commonly used inflexible chip materials .

As a further solution to the problem of gas bubbles inside a microfluidic device caused by the evaporation of the sample liquid, pressure can be applied to the microfluidic device can be exercised, as is the case, for example, in US 2005/0148066 A1 is described, in which a device for carrying out multiple simultaneous chemical and biochemical reactions in microvolumes in an array format is disclosed. Here the device is thermally cycled in a thermal cycler with a groove across the surface so that a thin microhole chip can be inserted into the groove and thermally cycled, where a thin thermally conductive silicone pad can be used to provide both thermal contact and pressure on the Surfaces of the microhole chip to prevent the evaporation of the samples within the array and thus the generation or growth of gas bubbles. A significant disadvantage of such a solution is the increased complexity of the entire instrument structure, as well as the additional structural complexity required by the application of pressure to the chip.

Therefore, in the technical field of dPCR testing, there is a general need to provide a microfluidic system and method for dPCR of a biological sample that has an improved solution for preventing and/or removing gas bubbles within the flow channel while thermocycling Efficiency of the microfluidic system is maintained or even improved.

SUMMARY OF THE INVENTION

The inventors of the present invention were able to confirm that gas bubbles can cause serious problems with regard to the successful completion of thermocycling of the samples, thus leading to significant assay failure. It has also turned out that the solutions proposed so far are not sufficient or satisfactory with regard to avoiding such gas bubbles. In particular, the solutions already proposed in the prior art (see above) were either too expensive or not sufficient to achieve reproducible test results. The introduction of gas bubbles into a flow channel of a microfluidic device as well as the formation of new gas bubbles and the growth of gas bubbles during temperature control must therefore be avoided, so that a new and improved solution became necessary. The inventors have developed a new and inventive solution, which is essentially based on the idea of removing existing or emerging gas bubbles by “rinsing” the microfluidic chip with separation liquid or sealing liquid, i.e. with separation liquid, since separation liquid - if they flows through the flow channel - can reliably flush any gas bubbles through the flow channel and out of the outlet opening of a microfluidic chip. The present invention thus solves the problems described above with a simplified and more effective avoidance and/or removal of gas bubbles in a flow channel of a microfluidic device within a microfluidic system while at the same time improving the efficiency of thermocycling.

According to a first aspect of the present invention, a microfluidic system for the dPCR of a biological sample is provided, which is used, for example, for examining a biological sample, which is provided in the form of a sample liquid, such as a polar aqueous sample solution, for individual reaction areas of an array of reaction areas wherein the microfluidic system comprises at least one microfluidic device having an inlet, an outlet, a flow channel connecting the inlet to the outlet, and an array of reaction regions in fluid communication with the flow channel. The microfluidic device can have a structure that consists of at least an upper layer and a lower layer, whereby either the upper layer or the lower layer can provide the arrangement of reaction areas, the inlet and the outlet. The flow channel is formed between the upper layer and the lower layer and is in fluid communication with the array of reaction regions, which may be in the form of microwells or nanowells, whereby the microfluidic device becomes, for example, a microfluidic chip. The width of the entire flow channel, also referred to as the path width, can, for example, be in a range between 6 mm and 7 mm, for example 6.4 mm, which usually provides space in width for approximately 60 to 100 wells next to each other, ie in one Transverse direction of the flow channel. In addition, the cross-sectional area of an opening of each depression may be circular, oval or polygonal, for example hexagonal, shaped. With a polygonal shape of the well opening and in particular with a hexagonal shape of the well opening, it is possible to arrange the well openings at a smaller distance from one another, ie to achieve a higher density of the distribution of the well openings in the flow channel. Accordingly, the number of wells or depressions in the arrangement of the wells of the plate can be further maximized. In addition, the width of the well opening, including a space between the wells, can be 60 µm ≤ w ≤ 110 µm, for example 62 µm (for a small well) ≤ w ≤ 104 µm (for a large well). Furthermore, the microfluidic system includes a flow circuit that can be connected to the microfluidic device in order to flow fluid liquid to flow through the flow channel of the microfluidic device, and a sample liquid source that can be connected to the microfluidic device to supply the microfluidic device with a sample liquid, for example by means of the flow circuit. Materials such as cyclic olefin copolymer (COC), cyclic olefin polymer (COP) or the like can be used as the material for the microfluidic device, ie for the device layers, with the use of COP being preferred, for example for cost reasons. Furthermore, the flow circuit can be realized by means of a hose system, for example a flexible hose system, consisting of one or more flexible hoses, the hoses consisting of an inner layer made of ethylene-propylene-diene monomer (EPDM) rubber and an outer layer made of nitrile butadiene (NBR) rubber, possibly also reinforced with a synthetic mesh, or generally made of EPDM, NBR, fluorinated ethylene-propylene polymer (FEP), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyethersulfone (PES), fluoroelastomer (FKM), silicone and can also be coated with a heat-insulating material.

In addition, the microfluidic system of the present invention includes a primary separation fluid source that can be connected to the microfluidic device to supply the microfluidic device with initial or primary separation fluid for sealing the sample fluid within the array of reaction regions, the initial separation fluid being a non-temperature separation fluid can be, i.e. a non-heated separating liquid which is provided, for example, at or approximately at ambient temperature. In addition, the microfluidic system of the present invention includes a secondary separation fluid source that can be connected to the microfluidic device to supply the microfluidic device with additional separation fluid, and a pumping means that is connected to the flow circuit and pumps the additional separation fluid through the flow channel can. The pumping means of the present invention can be, for example, a peristaltic pump, a metering pump or a syringe pump, or alternatively - with additional fluidic components such as valves or the like - the pumping means of the present invention can be any other type of pump, e.g. a diaphragm pump, a wobble piston pump, a micro gear pump or the like. Furthermore, as an example of the connectivity between the microfluidic device and the connectable components of the microfluidic system of the present invention, the inlet and/or the outlet of the microfluidic device may be designed in the form of a single connection, such as a circular fluid-tight connection, for For example, in the form of a Luer lock adapter or the like, for connecting the microfluidic device to the sample liquid source, for connecting the microfluidic device to the primary separation liquid source or for connecting the microfluidic device to the secondary separation liquid source.

With the above-described combination of separation liquid source and pumping device of the microfluidic system of the present invention and the possibility of pumping additional separation liquid through the flow channel, gas bubbles already present in the flow channel or any type of gas bubbles that arise during the thermocycling of the sample inside the microfluidic device arise can be removed from the microfluidic device by flushing the flow channel with additional separating liquid. Accordingly, with the present invention, the negative influence of gas bubbles on the test results can at least be reduced or completely avoided, resulting in a significantly improved microfluidic system.

In other words, the present invention addresses the general technical field of PCR using a well-plate based thermocycling structure, with emphasis on endpoint-aligned digital PCR. More specifically, the well plate is provided in a microfluidic chip in the form of a microtiter plate or even a nanowell plate with several thousand nanowells. This can be a disposable microfluidic chip in which the flow channel is in the form of a closed filling channel on the side of the recess openings, which on one side has the macroscopic inlet opening for the filling and on the other side with an outlet opening for connected to the overflow. The microfluidic disposable chip can also contain more than one array of reaction areas with such a microtiter plate structure and accordingly more than one filling channel on the side of the openings. As a rule, the microfluidic chip can be prefilled with a so-called PCR master mix, which is mixed with the target mixture in the form of the sample liquid. Subsequently, the flow channel on the side of the openings of the microwells is filled with a separating liquid, for example a cover oil, in order to prevent the sample liquid in the microwells from evaporating during thermocycling and the sample from migrating from well to well. Here the oil can be filled into the flow channel under a pressure of around 500 mbar, for example with the help of peristaltic pumps. Finally The microfluidic system of the present invention can be equipped with various sensors that check filling, correct placement and the like.

According to a specific embodiment of the microfluidic dPCR system of the present invention, the initial separation liquid and the additional separation liquid are made of one and the same separation liquid material, which means that each separation liquid used with the microfluidic system of the present invention can be the same type of separation liquid. In addition, each separating liquid must be immiscible with the sample liquid. As such an immiscible liquid, an oil liquid or a polymer liquid can be used, for example a silicone liquid such as polydimethylsiloxane (PDMS), or a mixture of an oil liquid and a polymer liquid. In addition, the initial separation liquid and the additional separation liquid can be provided by different separation liquid sources or alternatively from the same separation liquid source, i.e. the initial separation liquid source and the secondary separation liquid source can be realized by different separation liquid reservoirs or alternatively by a common separation liquid reservoir. In particular, in the event that the initial barrier liquid and the additional barrier liquid consist of one and the same separating liquid material or type, the primary separating liquid source and the secondary separating liquid source can be realized by a common separating liquid container, the terms “primary” and “secondary” being used in this case relate only to the functionality of the common separation liquid container, i.e. the common separation liquid container, i.e. the common separation liquid container can function as a primary separation liquid source for providing the initial separation liquid that primarily seals the sample within the array of reaction regions of the microfluidic device, and can then act as a secondary Separating liquid source for providing additional separating liquid for flushing out any gas bubbles from the flow channel of the microfluidic device. Such a configuration can be chosen because the complexity of the structure of the microfluidic system can be reduced compared to providing two different separation liquid containers.

According to a further specific embodiment of the dPCR microfluidic system of the present invention, the pumping device can be controlled by a control unit to pump the additional separation liquid through the flow channel when necessary to flush gas bubbles from the flow channel. The control unit can be triggered manually by an operator or automatically, for example based on a feedback signal from an additional system component that detects or monitors the occurrence of gas bubbles in the flow channel. Also, the control unit can instruct the pumping device to pump additional separation liquid through the flow channel in a predetermined pattern, for example based on the thermocycling steps to be applied to the sample liquid in the reaction areas. It can be advantageous that the pump device is controlled so that it pumps a predetermined amount of the additional separating liquid through the flow channel, whereby the pump device can be controlled so that it pumps the additional separating liquid either constantly, intermittently or when gas bubbles are detected in the flow channel pumps through the flow channel.

According to another specific embodiment of the dPCR microfluidic system of the present invention, the microfluidic system may further comprise a bubble trap connected to the flow circuit for separating air from the sealing liquid, the bubble trap being disposed downstream of the outlet of the microfluidic device. As a result, any gas bubbles that are flushed out of the flow channel of the microfluidic device by the additional separating liquid flowing out can be removed from the separating liquid flowing in the flow circuit. Accordingly, the optional bubble trap can be arranged in the fluid path of the separating liquid, as specified by the flow circuit, in order to separate air bubbles from the flowing separating liquid. As a result, gas bubbles can be immediately removed from the microfluidic system of the present invention without having to collect the separating liquid emerging from the microfluidic device and without having to extract gas bubbles therefrom.

According to a further specific embodiment of the dPCR microfluidic system of the present invention, the sample liquid may be an aqueous solution comprising the biological sample and the reagents required for the dPCR assay, wherein the sample liquid is first flowed through the flow channel into the arrangement of the reaction areas can be used to fill each reaction area with sample liquid. Subsequently, i.e. after providing the sample liquid for the arrangement of reaction regions, the initial separation liquid is flowed into the flow channel of the microfluidic device in order to seal the sample liquid within the reaction regions.

According to a further specific embodiment of the dPCR microfluidic system of the present invention, the microfluidic system may further comprise a detection device for detecting the presence or generation of gas bubbles in the microfluidic device, such as an optical imaging device, for example an optical camera, which detects the presence or can detect and/or monitor the generation of gas bubbles inside the flow channel before and during thermocycling, provided the flow channel enables optical monitoring. For example, the interior of the flow channel can be made visible from the outside, for example through a viewing window, transparent walls of the flow channel, or similar. Accordingly, the detection device can be used to provide a feedback signal that indicates the occurrence of gas bubbles inside the flow channel indicates, wherein the control unit can be triggered based on such a feedback signal. With such a structure, the microfluidic system can operate automatically without requiring an operator to monitor the microfluidic device during thermocycling or the like.

1. (a) die sekundäre Trennflüssigkeitsquelle ein Reservoir mit nicht beheizter zusätzlicher Trennflüssigkeit umfasst, wobei stromabwärts des Reservoirs eine Durchflusserwärmungseinrichtung vorgesehen ist, um die zusätzliche Trennflüssigkeit bei Bedarf zu erwärmen, d.h. um die durch die Durchflusserwärmungseinrichtung strömende Trennflüssigkeit zu erwärmen, wenn das Temperaturprofil des Thermozyklus dies erfordert, wobei die Durchflusserwärmungseinrichtung in Form einer Temperiereinrichtung, die um einen Teil des vom Trennflüssigkeitsreservoir kommenden Strömungskreislaufs vorgesehen ist, eines angeschlossenen Durchflusserhitzers oder dergleichen ausgeführt sein kann;
2. (b) die sekundäre Trennflüssigkeitsquelle mindestens ein Reservoir mit erwärmter zusätzlicher Trennflüssigkeit und mindestens ein Reservoir mit nicht erwärmter oder gekühlter zusätzlicher Trennflüssigkeit umfasst, wobei die Reservoire mittels eines jeweiligen Ventilmechanismus oder dergleichen, wie beispielsweise eines elektronisch gesteuerten Abgabeventils, trennbar mit dem Strömungskreislauf verbunden sein können, oder die Reservoire mittels eines Mischventils mit dem Strömungskreislauf verbunden sein können, um erwärmte zusätzliche Trennflüssigkeit und nicht erwärmte oder gekühlte zusätzliche Trennflüssigkeit zu mischen, um die gewünschte Temperatur der der mikrofluidischen Vorrichtung zuzuführenden Trennflüssigkeit zu erreichen;
3. (c) die zusätzliche Trennflüssigkeit ein elektrisch leitfähiges Material, wie z.B. Graphen, umfasst, um die zusätzliche Trennflüssigkeit bei Bedarf durch die Anwendung von Elektrizität auf die zusätzliche Trennflüssigkeit zu erhitzen.

To provide the sample material in the reaction regions with a thermal cycling temperature profile, the microfluidic system may include a thermal holder that receives the microfluidic device to provide the array of reaction regions with a thermal cycling temperature profile. For example, a thermal structure such as that associated with one of the 4A until 4D described, that is, a so-called plate cycler can accommodate the microfluidic device of the microfluidic system of the present invention. Alternatively, a thermocycling temperature profile for the array of reaction regions can be provided by means of a heating and/or cooling device for heating and/or cooling the temperature of the additional separating liquid to a desired thermocycling temperature profile temperature. Thus, the desired thermocycling temperature profile of the contents of the reaction regions can be realized not by supplying heat by means of an external heat source in thermal connection with the microfluidic device, but by means of a respectively heated or cooled separating liquid that flows through the flow channel of the microfluidic device, ie a new type the supply of heat to the sample material, whereby any type of gas bubbles in the flow channel can be flushed out. With such a solution, not only the removal of gas bubbles, but also the heating and/or cooling of the biological sample in the reaction areas within the microfluidic device can be achieved. Accordingly, a more direct and rapid application of a thermocycling temperature profile to the biological samples can also be achieved, wherein the commonly known external components, such as an upper heater and a lower heater, can be omitted, thereby simplifying the known structure of a thermocycling device . To achieve such temperature control from within the microfluidic device instead of external heat input, there are several options that can be combined if desired, including the following:

1. (a) the secondary separation liquid source comprises a reservoir with unheated additional separation liquid, a flow-through heating device being provided downstream of the reservoir in order to heat the additional separation liquid if necessary, ie to heat the separation liquid flowing through the flow-through heating device if the temperature profile of the thermal cycle so requires, wherein the flow heating device can be designed in the form of a temperature control device, which is provided around a part of the flow circuit coming from the separating liquid reservoir, a connected flow heater or the like;
2. (b) the secondary separation liquid source comprises at least one reservoir with heated additional separation liquid and at least one reservoir with unheated or cooled additional separation liquid, wherein the reservoirs can be separably connected to the flow circuit by means of a respective valve mechanism or the like, such as an electronically controlled dispensing valve , or the reservoirs can be connected to the flow circuit by means of a mixing valve in order to mix heated additional separation liquid and non-heated or cooled additional separation liquid in order to achieve the desired temperature of the separation liquid to be supplied to the microfluidic device;
3. (c) the additional separation fluid comprises an electrically conductive material, such as graphene, to heat the additional separation fluid as needed by applying electricity to the additional separation fluid.

When using one of options (a) and (b) or a combination thereof, the pumping device must be adapted so that it can follow the desired dPCR cycles, ie bring the respective tempered additional separation liquid over and away from the sample array and any Amount of additional separating fluid Pump through the flow channel to remove gas bubbles. In the case of option (c), i.e. when the separation fluid is electrically heated, it should be an electrically conductive fluid that provides suitable resistance so that it heats up when current flows. A graphene solution is suitable for such an application. In this case, however, the separating liquid cannot be cooled electrically; instead, either the reservoir or the microfluidic chip itself must be cooled if the sample temperature needs to be lowered during dPCR. Furthermore, when using the electric heating solution, a circular separation fluid system without a reservoir could be used, which would require precise control of the dPCR cycles, for example through a comprehensive sensor structure that monitors the temperatures and flows in real time.

According to a further embodiment of the dPCR microfluidic system of the present invention, the microfluidic system may additionally comprise a pressure chamber that surrounds at least the microfluidic device. Here, the functionality of such an additional pressure chamber can complement the already achieved avoidance or elimination of gas bubbles inside the microfluidic system of the present invention and thus further ensure that gas bubbles cannot interfere with the assay results. For the thermal cycling itself, the microfluidic disposable device including the oil-filled openings can therefore be placed under a pressure of 1 to 2 bar, for example about 1.5 bar, in order to further suppress the formation of bubbles during thermal cycling. The pressure is preferably applied to the inside of the microfluidic device by arranging the entire microfluidic device within the pressure chamber.

According to a further specific embodiment of the present invention, the device according to the invention can further comprise at least one sensor for monitoring the temperature of the biological samples recorded in the microfluidic device, such a sensor being a temperature sensor, for example in combination with a fluid flow sensor. By providing a corresponding sensor on one of the components of the microfluidic system according to the invention, the temperature of the biological samples can be accurately monitored during dPCR, and the heating/cooling function of the microfluidic system can be controlled based on the measured temperature values of the samples in order to achieve the various To regulate temperature plateaus of dPCR precisely and efficiently. Accordingly, such sensors, e.g., temperature sensors, enable precise control of the thermocycling temperature through a control algorithm or the like, using temperature sensors and other sensors to control the respective heating/cooling rate, with the heating/cooling output being varied significantly by changing the fluid pumping speed can.

Furthermore, a method for dPCR of a biological sample in a microfluidic system as described above is described herein, the method comprising a step of flowing a sample liquid through the flow channel of the microfluidic device, which is provided in particular in the form of a microfluidic chip, and filling the arrangement of reaction regions with the sample liquid in a sequential manner by forcing the sample liquid through the flow channel, a subsequent step of flowing an initial separation liquid through the flow channel of the microfluidic device to seal each reaction region after it has been filled with the sample liquid, and remaining squeezing sample liquid out of the microfluidic device, a step of applying a thermocycling temperature profile to the array of reaction regions, and a step of pumping additional separation liquid through the flow channel of the microfluidic device to flush gas bubbles from the flow channel. The step of applying a temperature profile to the arrangement of reaction areas and the step of pumping additional separating liquid through the flow channel of the microfluidic device for flushing out gas bubbles from the flow channel can take place in a combined step, i.e. if the array of reaction areas is exposed to a Temperature is achieved by pumping heated additional separation liquid through the flow channel, the pumping of the additional separation liquid leading to a heating of the sample liquid inside the reaction array and at the same time to the flushing out of gas bubbles from the flow channel. More preferably, the dPCR method described also includes a step of assaying the biological sample provided in the array of reaction areas, whereby the method becomes a dPCR-based analysis method.

The step of pumping additional separation fluid through the flow channel may include pumping a predetermined amount of additional separation fluid through the flow channel as needed, wherein the step of pumping additional separation fluid through the flow channel is either a step of continuously pumping additional separation fluid through the flow channel or a step of intermittently pumping additional separating liquid through the flow channel, for example only if the sample liquid has to be tempered within the reaction areas, and / or can be a step of pumping additional separating liquid through the flow channel when gas bubbles are detected in the flow channel. Due to the possibility of pumping additional separating liquid through the flow channel if necessary, existing gas bubbles in the flow channel or any type of gas bubbles that arise during the thermocycling of the sample inside the microfluidic device can be removed by flushing the flow channel with additional separating liquid from the microfluidic device be removed. Accordingly, with this method, the negative influence of gas bubbles on the test results can at least be reduced or completely avoided, which leads to a significantly improved method for dPCR of a biological sample in the microfluidic system according to the invention.

The method described herein may further comprise a step of monitoring and detecting the presence or generation of gas bubbles in the microfluidic device, for example using the detection means, for example in the form of an optical camera or the like, which detects the presence or generation of gas bubbles in the microfluidic device The interior of the flow channel can be detected and/or monitored before and during thermal cycling, provided that the flow channel enables optical monitoring, i.e. the interior of the flow channel is visible from the outside, for example by means of a viewing window, transparent walls of the flow channel or the like. Accordingly, the detection device can be used to provide a feedback signal indicating the occurrence of gas bubbles inside the flow channel, wherein the control unit can be triggered based on such feedback signal, resulting in a feedback-controlled dPCR method. Alternatively or additionally, the method may include a further step for separating gas bubbles from the additional separation liquid, for example with the aid of the bubble trap, which is connected to the flow circuit downstream of the outlet of the microfluidic device. In this way, every gas bubble that is flushed out of the flow channel of the microfluidic device by the additional separating liquid flowing can be removed from the separating liquid flowing in the flow circuit. Accordingly, the optional step of separating gas bubbles from the additional separation liquid using the bubble trap disposed in the fluid path of the separation liquid may be useful to immediately remove gas bubbles from the microfluidic system of the present invention without the need to collect the separation liquid exiting the microfluidic device and extract any gas bubbles from it at a later date. Alternatively or additionally, the method can also include a step of pressurizing the microfluidic device, for example by means of a pressure chamber that surrounds at least the microfluidic device. The functionality of such an additional pressure application step can complement the already achieved avoidance or removal capability of the dPCR method, further ensuring that gas bubbles do not interfere with test results. The microfluidic device can be pressurized with a pressure of 1 to 2 bar, for example about 1.5 bar, in order to further suppress the formation of bubbles during thermocycling, the pressure being able to be applied to the interior of the microfluidic device, either by pressurization a side wall of the flow channel, which acts as a flexible pressure-transmitting component, or by pressurizing the entire microfluidic device, which in this case must have a certain compressibility.

1. (a) einen Schritt zur Steuerung einer stromabwärts von der sekundären Trennflüssigkeitsquelle vorgesehenen Durchflussheizeinrichtung, um die zusätzliche Trennflüssigkeit bei Bedarf zu erwärmen;
2. (b) einen Schritt der Steuerung eines Ventilmechanismus zum Mischen von zusätzlicher Trennflüssigkeit aus mindestens einem Vorratsbehälter mit einer beheizten zusätzlichen Trennflüssigkeit und mindestens einem Vorratsbehälter mit einer nicht beheizten oder gekühlten zusätzlichen Trennflüssigkeit;
3. (c) einen Schritt des Anlegens von Elektrizität an die zusätzliche Trennflüssigkeit je nach Bedarf, wobei die zusätzliche Trennflüssigkeit elektrisch leitendes Material, wie Graphen-Material, zur Erwärmung der zusätzlichen Trennflüssigkeit durch die Anwendung von Elektrizität umfasst;
4. (d) eine Kombination der zuvor genannten optionalen Schritte (a) bis (c).

The step of applying a thermocycling temperature profile to the array of reaction regions may include a step of controlling the temperature profile of a thermal fixture that houses the microfluidic device to provide a thermocycling temperature profile to the array of reaction regions, or, alternatively, a step controlling a heating and/or cooling device for heating and/or cooling the temperature of the separating liquid to a desired thermocycling temperature profile temperature. The step of controlling a heating and/or cooling device can include one of the following options:

1. (a) a step of controlling a flow heater provided downstream of the secondary separation fluid source to heat the additional separation fluid as needed;
2. (b) a step of controlling a valve mechanism for mixing additional separation liquid from at least one storage container with a heated additional separation liquid and at least one storage container with a non-heated or cooled additional separation liquid;
3. (c) a step of applying electricity to the additional separation liquid as needed, the additional separation liquid comprising electrically conductive material, such as graphene material, for heating the additional separation liquid by the application of electricity;
4. (d) a combination of the aforementioned optional steps (a) to (c).

The microfluidic system of the present invention described above may be part of an automated processing system, such as an analytical, pre-analytical or post-analytical processing system commonly used in modern laboratories for automatic processing of biological samples, which may include any device or device component which which is/are capable of carrying out one or more processing steps/operations on one or more biological samples, and which includes analytical instruments, pre-analytical instruments and also post-analytical instruments. The term “processing steps” refers to physically carried out processing steps, such as carrying out the individual steps of a dPCR process. The term “analytical,” as used herein, includes any process step performed by one or more laboratory instruments or operational units capable of performing an analytical test on one or more biological samples. In the context of biomedical research, analytical processing is a technical procedure for characterizing the parameters of a biological sample or analyte. Such characterization of parameters includes, for example, the determination of the concentration of certain proteins, nucleic acids, metabolites, ions or molecules of various sizes in biological samples derived from humans or laboratory animals, or the like. The information collected can be used, for example, to evaluate the effects of drug administration on the organism or on certain tissues. Further analyzes can determine optical, electrochemical or other parameters of the biological samples or the analytes contained in the sample material.

The method steps described above can be controlled by the control unit of the described microfluidic system, which can also control any type of actuation or monitoring of the above-described microfluidic system and its components, where the term “control unit” as used herein means any physical or virtual processing device, such as a CPU or the like, which can also control an entire laboratory instrument or even an entire workstation comprising one or more laboratory instruments so that workflows and work steps are carried out. For example, the control unit may carry various types of application software and instruct the automated processing system or a specific instrument or device thereof to perform pre-analytical, post-analytical and analytical workflows/operations. The control unit can receive information from a data management unit about which steps need to be carried out with a particular sample. In addition, the control unit can be integrated into a data management unit, consist of a server computer and/or be part of an instrument or even be distributed across several instruments of the automated processing system. The control unit can, for example, be implemented as a programmable logic controller that executes a computer-readable program with instructions for carrying out operations. In order to receive such instructions from a user, a user interface may additionally be provided, where the term "user interface" as used herein includes any suitable application software and/or hardware for interaction between an operator and a machine, including but not limited to not limited to a graphical user interface that receives as input a command from an operator and also provides feedback and information to the operator. A system/device may also have multiple user interfaces to serve different types of users/operators.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include the plural unless the context clearly requires otherwise. Similarly, the words "include", "include" and "include" are to be understood inclusively rather than exclusively, i.e. in the sense of "including but not limited to". Likewise, the word “or” shall include “and” unless the context clearly indicates otherwise. The terms "multiplicity", "plurality" or "plurality" refer to two or more, i.e. 2 or >2, with integer multiples, while the terms "singular" or "singularity" refer to one, i.e. =1. In addition, the term “at least one” is to be understood as one or more, i.e. 1 or >1, including integer multiples. Accordingly, words that use the singular or plural also include the plural or singular. In addition, the words “herein,” “above,” “before,” and “below,” and words of similar meaning, when used in this application, refer to this application as a whole and not to any particular portion of the application.

The description of specific embodiments of the present invention is not intended to be complete or to limit the disclosure to the precise form stated. While the specific embodiments and examples of the present disclosure are here Illustratively, various equivalent modifications are possible within the scope of the disclosure as set forth in the appended claims, as will be apparent to one skilled in the art. Certain elements of the embodiments described above and later may be combined or replaced with elements in other embodiments. To avoid repetition, the same elements are designated by the same reference numerals in the drawings, and parts that are easily implemented by one skilled in the art may be omitted. While the advantages associated with certain embodiments of the disclosure have been described in connection with these embodiments, other embodiments may also have such advantages, and not all embodiments must necessarily have such advantages to fall within the scope of the disclosure as defined by the appended claims.

The following examples are provided to illustrate specific embodiments of the present invention. The specific embodiments described below should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various alterations, changes and modifications may be made without departing from the scope of the present invention as defined by the appended claims, and it is therefore to be understood that such equivalent embodiments are included herein. Further aspects and advantages of the present invention will become apparent from the following description of the embodiments shown in the figures.

BRIEF DESCRIPTION OF DRAWINGS

• 1 is a cross-sectional conceptual representation of a microfluidic system for dPCR of a biological sample according to a first embodiment of the present invention;
• 2 is a cross-sectional conceptual representation of a microfluidic system for dPCR of a biological sample according to a second embodiment of the present invention;
• 3A and 3B are schematic structural representations of a known microfluidic system in plan view and cross-sectional view; and
• 4A until 4D show different stages of a dPCR with schematic functional representations of the growth and generation of gas bubbles in a known microfluidic system.

LIST OF REFERENCE NUMBERS

1

microfluidic system (first embodiment)

1'

microfluidic system (second embodiment)

2

microfluidic device

21

bottom plate/bottom layer of the microfluidic device

22

top plate/top layer of the microfluidic device

23

Inlet of the microfluidic device

231

Microfluidic device inlet cover

24

Microfluidic device outlet

241

Microfluidic device outlet cover

25

Flow channel of the microfluidic device

26

Arrangement/array of reaction areas/wells/wells

27

Sample liquid (filled into the reaction areas)

28

initial separation fluid

29

additional separating fluid

3

Flow circuit

31

pump

32

Bubble trap

4

secondary separation fluid source

41

tempered reservoir

411

electronically controlled delivery valve

42

untempered reservoir

421

electronically controlled delivery valve

5

Instantaneous water heater

6

Microfluidic chip / microfluidic chip

61

bottom plate/bottom layer of the microfluidic chip

62

top plate / top layer of the microfluidic chip

63

Inlet of the microfluidic chip

64

Microfluidic chip outlet

65

Flow channel of the microfluidic chip

66

Array of microwells/microwells

7

Microfluidic chip / microfluidic chip

71

bottom plate/bottom layer of the microfluidic chip

72

top plate / top layer of the microfluidic chip

73

Inlet of the microfluidic chip

74

Microfluidic chip outlet

75

Flow channel of the microfluidic chip

76

Arrangement of the reaction areas / depressions / wells

77

Sample liquid (filled into the reaction areas)

78

separating fluid

8th

Plate cycler

81

lower heater of the plate cycler

82

plate cycle upper heater

91

initial gas bubble

92

newly emerging gas bubble

93

fused gas bubble

DETAILED DESCRIPTION

1 shows a microfluidic system 1 for dPCR of a biological sample according to a first embodiment of the present invention in a schematic representation, the main components being shown either in an illustrative cross section or as a pictogram. This in 1 The microfluidic system 1 shown includes a microfluidic device 2 in the form of a microfluidic chip, the device 2 having a similar structure to that in 3B chip 7 shown from the prior art, wherein the device 2 essentially consists of a lower plate 21 and an upper plate 22 and has an inlet 23, an outlet 24 and a flow channel 25 which connects the inlet 23 to the outlet 24 . Furthermore, an arrangement of reaction areas 26 in the form of microwells or nanowells is provided in the flow channel 25 on its upper side, ie on the inside of the upper plate 22, in order to be able to monitor the reactions in the reaction areas 26 from above. In addition, the microfluidic device 2 in the state shown has already been rinsed with sample liquid 27, which is filled into the arrangement of the reaction areas 26, ie which is to be filled into each individual microwell of the arrangement of the reaction areas 26. Such filling of the sample liquid 27 into the array of reaction regions 26 can be carried out at a filling station (not shown) before the microfluidic device 2 is arranged within the microfluidic system 1, the filling station (not shown) being considered as part of the microfluidic system 1 which is used for introducing the sample liquid 27 into the array of reaction areas 26. Also, the initial separation liquid 28 has already been filled into the microfluidic device 2, which seals the sample liquid 27 within the arrangement of reaction regions 26, the filling of the initial separation liquid 28 into the flow channel 25 in order to seal the arrangement of reaction regions 26 also on the filling station (not shown) can be made before the microfluidic device 2 is arranged within the microfluidic system 1. Alternatively, the initial separation liquid 28 can be filled into the flow channel 25 to close the array of reaction regions 26 by means of the microfluidic system 1 itself, namely by means of the additional separation liquid 29, the part of the additional separation liquid 29 which contains the sample liquid 29 within the Arrays of reaction areas 26 actually closes, is to be understood as “initial” separating liquid 28. Since here the filling station can be connected to the microfluidic device 2 in order to supply the microfluidic device 2 with sample liquid 27, the filling station (not shown) can be viewed as a sample liquid source and as a primary separation liquid source, as a connectable combined functional part of the microfluidic system 1.

The microfluidic system 1 further comprises a flow circuit 3, which is connected to the microfluidic device 2, the flow circuit 3 serving to guide additional separating liquid 29 through the flow channel 25 of the microfluidic device 2, primarily to pass the additional separating liquid 29 to guide the flow channel 25 of the microfluidic device 2. The flow of the additional separating liquid 29 is shown here by an arrow circle rotating counterclockwise. The additional separating liquid 29 essentially comes from a secondary separating liquid source 4, which consists of a temperature-controlled reservoir 41 with heated additional separating liquid 29 and a non-tempered reservoir 42 with non-heated or cooled additional separating liquid 29, the reservoirs 41, 42 each having an electronic controlled dispensing valve 411, 421 are connected to the flow circuit 3. Thus, heated additional separating liquid 29 can be introduced into the flow circuit 3 by controlling the delivery valve 411 and unheated additional separating liquid 29 can be introduced into the flow circuit 3 by controlling the delivery valve 421, which leads to a desired temperature profile of the additional separating liquid 29 in the flow circuit 3. Alternatively, the tempered reservoir 41 and the untempered reservoir 42 can share a common mixing valve, which is then connected to the flow circuit 3 in the sense of a mixing tap, so that already pre-mixed and therefore correspondingly tempered additional separating liquid 29 is introduced into the flow circuit 3 and the microfluidic Device 2 is supplied.

In order to supply the additional separating liquid 29 to the flow channel 25, a peristaltic pump 31, which functions as a pumping device of the microfluidic system 1, is provided as part of the flow circuit 3 in order to supply the additional separating liquid 29 through its inlet 23 through the flow channel 25 and out of the outlet 24 to the microfluidic device 2. This not only supplies the arrangement of the reaction areas 26 with heat or cold in order to thermocycle the samples within the arrangement of the reaction areas 26, but it is also achieved that gas bubbles occur within the flow channel 25 , from the flow channel 25 and the outlet 24, i.e. from the microfluidic device 2 are flushed out. The pump 31 can constantly pump the additional separating liquid 29 through the flow channel 25, or it can pump the additional separating liquid 29 intermittently through the flow channel 25, for example only when the sample liquid 27 needs to be tempered within the arrangement of reaction areas 26. Due to the possibility of pumping additional separating liquid 29 through the flow channel 25 if necessary, existing gas bubbles within the flow channel 25 or any type of gas bubbles that arise during thermocycling of the sample liquid 27 can be removed by flushing the flow channel 25 with additional separating liquid 29 microfluidic device 2 can be removed. In order to be able to finally remove any gas bubbles from the entire flow system, the microfluidic system 1 further comprises a bubble trap 32 arranged downstream of the outlet 24 of the microfluidic device 2 and in the present embodiment in front of the pump 31, with which any gas bubbles are removed from the additional separating liquid 29 that flows be separated. In this way, any gas bubbles can be immediately removed from the microfluidic system 1. This means that a negative influence of gas bubbles on the test results can at least be reduced or avoided entirely.

In 2 1 shows a second embodiment of a microfluidic device 1' of the present invention, which essentially consists of a similar structure to that in relation to the first embodiment in FIG 1 is described. Here, like reference numerals denote like elements, and the respective descriptions thereof are omitted to avoid repetition. In contrast to the first embodiment, however, the microfluidic device 1 'comprises only the untempered reservoir 42, which contains an unheated additional separation liquid 29, with a flow heater 5 being provided downstream of the reservoir 42 in order to heat the untempered reservoir 42 according to a desired thermocycling process. Temperature profile to be applied to the samples within the arrangement of reaction areas 26. Here too, the reservoir 42 is connected to the flow circuit 3 via a corresponding electronically controlled delivery valve 421, whereby an appropriately tempered additional separating liquid 29 can be introduced into the flow circuit 3 by controlling the delivery valve 421 and pumped into the microfluidic device 2 by the pump 31 not only to apply the desired thermocycling temperature profile to the samples within the array of reaction areas 26, but above all also to flush out any gas bubbles that may appear in the flow channel 25.

According to an alternative embodiment, the structure of the previously described embodiment as shown in 2 shown, but without the flow heater 5, can be applied to a plate cycler 8, as is known and in connection with one of the 4A until 4D described, wherein the plate cycler 8 has the microfluidic device 2 provided therein. In this way, the plate cycler 8 can impart a desired thermocycling temperature profile to the sample liquid 27 in the arrangement of the reaction regions 26, and the pumping of the unheated additional separation liquid 29 is only used to remove gas bubbles in the flow channel 25, if present. Furthermore, according to another alternative embodiment, the structure of the in 2 shown embodiment without a flow heater 5 or without a plate cycler 8 can be used, however, the additional separation liquid 29 comprises electrically conductive graphene material to heat the additional separation liquid 29 by the application of electricity to the additional separation liquid 29 when necessary, thereby heating the additional Separating fluid 29 is achieved in order to able to apply a desired thermocycling temperature profile to the sample material within the array of reaction regions 26 without requiring any type of external heater or heated reservoir.

Although the present invention has been described with respect to its specific embodiments, this description is for illustrative purposes only. Accordingly, the invention is limited only by the scope of the appended claims.

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• EP 2830769 A1 [0009]
• US 20050009101 A1 [0010]
• US 20050148066 A1 [0011]

Claims (65)

Microfluidic system (1; 1') for the digital polymerase chain reaction dPCR of a biological sample, the microfluidic system (1; 1') comprising: at least one microfluidic device (2) with an inlet (23), an outlet (24), a flow channel (25) which is arranged between the inlet (23) and the outlet (24) and the inlet (23) with the outlet (24) connects, and an arrangement of reaction areas (26) which are in fluid communication with the flow channel (25); a flow circuit (3) connectable to the microfluidic device (2) to allow liquid to flow through the flow channel (25) of the microfluidic device (2); a sample liquid source which can be connected to the microfluidic device (2) in order to supply the flow channel (25) and thus the arrangement of reaction regions (26) of the microfluidic device (2) with a sample liquid (27) via the inlet (23); a primary separation liquid source connectable to the microfluidic device (2) to supply the flow channel of the microfluidic device (2) via the inlet (23) with initial separation liquid (28) for sealing the sample liquid (27) within the array of reaction regions (26 ) to supply; a secondary separation liquid source (4) which can be connected to the microfluidic device (2) in order to supply the flow channel (25) of the microfluidic device (2) with additional separation liquid (29) via the inlet (23), a pump device (31) which is connected to the flow circuit (3) and is suitable for pumping the additional separating liquid (29) through the flow channel (25), and a control device in the form of a programmable logic controller for executing a computer-readable program with instructions for carrying out operations, which is set up to control the pumping device (31) in order to pump the additional separating liquid (29) through the flow channel (25) if necessary, to flush gas bubbles from the flow channel (25). Microfluidic system (1; 1') according to Protection claim 1 , wherein the sample liquid (27) is an aqueous solution comprising the biological sample and the reagents required for the dPCR assay, and wherein each separation liquid (28, 29) is a liquid that is immiscible with the sample liquid (27). Microfluidic system (1; 1') according to Protection claim 2 , wherein each separation liquid (28, 29) is either oil or a polymer liquid, such as a silicone liquid, or a mixture thereof. Microfluidic system (1; 1') according to Protection claim 3 , wherein the initial separation liquid (28) and the additional separation liquid (29) consist of the same separation liquid material. Microfluidic system (1; 1') according to one of the preceding claims, wherein the primary separation liquid source and the secondary separation liquid source (4) are realized by different separation liquid reservoirs or by a common separation liquid reservoir. Microfluidic system (1; 1') according to Protection claim 5 , whereby the initial separating liquid (28) is an untempered separating liquid. Microfluidic system (1; 1') according to Protection claim 1 , wherein the pump device (31) is controlled so that it pumps a predetermined amount of the additional separation liquid (29) through the flow channel (25). Microfluidic system (1; 1') according to Protection claim 7 , wherein the pump device (31) is controlled in such a way that it pumps the additional separating liquid (29) through the flow channel (25) constantly, intermittently or when gas bubbles are detected in the flow channel (25). Microfluidic system (1; 1') according to one of the preceding claims, wherein the microfluidic system (1; 1') further has a bubble trap (32) connected to the flow circuit (3) for separating gas from the separating liquid (28, 29). , wherein the bubble trap (32) is arranged downstream of the outlet (24) of the microfluidic device (2). Microfluidic system (1; 1') according to one of the preceding claims, wherein the microfluidic system (1; 1') is used to examine the biological sample which is supplied in the form of the sample liquid (27) to each element of the array of reaction areas (26). provided. Microfluidic system (1; 1') according to one of the preceding claims, wherein the inlet (23) and/or the outlet (24) are in the form of a connection port for connecting the microfluidic device (2) to the sample liquid source, for connecting the microfluidic device ( 2) are designed with the primary separation liquid source and/or for connecting the microfluidic device (2) to the secondary separation liquid source (4). Microfluidic system (1; 1') according to one of the preceding claims, wherein the microfluidic device (2) is constructed with at least one lower layer (21) and an upper layer (22), either the lower layer (21) or the upper Layer (22) provides the array of reaction areas (26), the inlet (23) and the outlet (24), and wherein the flow channel (25) is set up between the lower layer (21) and the upper layer (22) and in There is fluid connection to the array of reaction areas (26). Microfluidic system (1; 1') according to Protection claim 12 , where each reaction region is a microwell or a nanowell. Microfluidic system (1; 1') according to Protection claim 13 , wherein the microfluidic device (2) is a microfluidic chip Microfluidic system (1; 1') according to one of the preceding claims, wherein the sample liquid (27) is an aqueous solution which contains the biological sample and reagents for the dPCR assay. Microfluidic system (1; 1') according to Protection claim 15 , wherein first the sample liquid (27) is flowed through the flow channel (25) into the arrangement of reaction areas (26), and wherein the initial separation liquid (28) is flowed into the flow channel (25) of the microfluidic device (2) after the Sample liquid (27) was supplied to the arrangement of reaction areas (26). Microfluidic system (1; 1') according to one of the preceding claims, wherein the microfluidic system (1; 1') further comprises a detection means for detecting the presence or generation of gas bubbles in the microfluidic device (2). Microfluidic system (1; 1') according to Protection claim 17 , wherein the detection means is an optical imaging device. Microfluidic system (1; 1') according to Protection claim 18 , where the detection means is an optical camera. Microfluidic system (1; 1') according to one of the preceding protection claims, wherein the microfluidic system (1; 1') further comprises a thermal holder which accommodates the microfluidic device (2) in order to provide the array of reaction regions (26) with thermocycling. To give temperature profile. Microfluidic system (1; 1') according to one of Protection claims 1 until 19 , wherein provision of a thermocycling temperature profile for the arrangement of reaction areas (26) is implemented by means of a heating and/or cooling device for heating and/or cooling the temperature of the separating liquid to a desired thermocycling temperature profile temperature. Microfluidic system (1; 1') according to Protection claim 21 , wherein the secondary separation liquid source (4) comprises a storage container (42) with unheated additional separation liquid (29), a flow heating device (5) being provided downstream of the storage container (42) in order to heat the additional separation liquid (29) if necessary. Microfluidic system (1; 1') according to Protection claim 21 or 22 , wherein the secondary separation liquid source (4) comprises at least one reservoir (41) with heated additional separation liquid (29) and at least one reservoir (42) with non-heated or cooled additional separation liquid (29), the reservoirs (41, 42) being separated by a Valve mechanism (411, 421), such as a respective electronically controlled dispensing valve (411, 421) or a common mixing valve, are separably connected to the flow circuit (3). Microfluidic system (1; 1') according to one of Protection claims 21 until 23 , the additional separating liquid (29) being electrically conductive material, such as. B. graphene material, to heat the additional separation liquid (29) by applying electricity to the additional separation liquid if necessary. Microfluidic system (1; 1') according to one of the preceding claims, wherein the microfluidic system (1; 1') further comprises a pressure chamber which surrounds at least the microfluidic device (2). Use of a microfluidic system (1; 1') according to one of the preceding claims for flushing out gas bubbles from the flow channel (25) by means of additional separating liquid (29) after the sample liquid (27) within the arrangement of reaction areas (26) by initial separating liquid (28 ) has been completely sealed. Use of a microfluidic system (1; 1'). Protection claim 26 , wherein sample liquid (27) is flowed through the flow channel (25) of the microfluidic device (2), which can be provided in the form of a microfluidic chip, and the arrangement of reaction areas (26) successively by pushing the Sample liquid (27) is filled with the sample liquid (27) through the flow channel (25); initial sealing liquid (28) is flowed through the flow channel (25) of the microfluidic device (2), for sealing each reaction region of the arrangement of reaction regions (26) after it has been filled with the sample liquid (27), and for squeezing out the remaining sample liquid ( 27) from the microfluidic device (2); a thermocycling temperature profile is applied to the array of reaction regions (26), and additional separation liquid (29) is pumped through the flow channel (25) of the microfluidic device (2) in order to flush gas bubbles from the flow channel (25). Use of a microfluidic system (1; 1'). Protection claim 26 or 27 , wherein the additional separation liquid (29) is pumped through the flow channel (25) after sealing the sample liquid (27) within the arrangement of reaction regions (26) by initial separation liquid (28), the pumping being pumping a predetermined amount of additional separation liquid (29) through the flow channel (25) on request. Use of a microfluidic system (1; 1'). Protection claim 28 , wherein the pumping of additional separating liquid (29) through the flow channel (25) involves constant pumping of additional separating liquid (29) through the flow channel (25) or intermittent pumping of additional separating liquid (29) through the flow channel (25) or pumping of additional separating liquid (29) through the flow channel (25) when gas bubbles are detected in the flow channel (25). Use of a microfluidic system (1; 1') according to one of Protection claims 26 until 29 , wherein the presence or generation of gas bubbles in the microfluidic device (2) is monitored and detected. Use of a microfluidic system (1; 1'). Protection claim 30 , wherein the presence or generation of gas bubbles in the microfluidic device (2) is monitored and detected by means of a detection means, such as an optical imaging device, for example an optical camera. Use of a microfluidic system (1; 1') according to one of Protection claims 26 until 31 , whereby gas bubbles are separated from the additional separating liquid (29). Use of a microfluidic system (1; 1'). Protection claim 32 , the separation taking place by means of a bubble trap (32) which is connected to the flow circuit (3) downstream of the outlet (24) of the microfluidic device (2). Use of a microfluidic system (1; 1') according to one of Protection claims 26 until 33 , whereby pressure is applied to the microfluidic device (2). Use of a microfluidic system (1; 1'). Protection claim 34 , wherein the pressure is applied by means of a pressure chamber which surrounds at least the microfluidic device (2). Use of a microfluidic system (1; 1') according to one of Protection claims 26 until 35 , wherein the biological sample provided in the array of reaction areas (26) is tested. Use of a microfluidic system (1; 1') according to one of Protection claims 26 until 36 , wherein applying the thermocycling temperature profile to the array of reaction regions (26) includes controlling the temperature profile of a thermal holder that accommodates the microfluidic device (2) to provide a thermocycling temperature profile to the array of reaction regions (26), or a control of a heating device for heating or a cooling device for cooling the temperature of the separation liquid to a desired temperature of the thermocycling temperature profile, or a control of a heating device for heating and a cooling device for cooling the temperature of the separation liquid to a desired temperature of the thermocycling temperature profile . Use of a microfluidic system (1; 1'). Protection claim 37 , wherein the control of the heating or cooling device (5) includes controlling a flow-through heating device (5) provided downstream of the secondary separation liquid source (4) for heating the additional separation liquid (29) on demand. Use of a microfluidic system (1; 1') according to one of Protection claims 37 and 38 , wherein the control of the heating or cooling device (5) involves controlling a valve mechanism (411, 421) for mixing additional separation liquid (29) from at least one reservoir (41) with a heated additional separation liquid (29) and at least one reservoir (42 ) with one not heated or cooled additional separating liquid (29). Use of a microfluidic system (1; 1') according to one of Protection claims 37 until 39 , wherein the control of the heating or cooling device (5) comprises applying electricity to the additional separating liquid (29) on demand, the additional separating liquid (29) passing through electrically conductive material, such as graphene material, for heating the additional separating liquid (29). includes the creation of electricity. Microfluidic system (1; 1') for the digital polymerase chain reaction dPCR of a biological sample, the microfluidic system (1; 1') comprising: at least one microfluidic device (2) with an inlet (23), an outlet (24), a flow channel (25) which connects the inlet (23) to the outlet (24), and an arrangement of reaction areas (26), which be in fluid communication with the flow channel (25); a flow circuit (3) connectable to the microfluidic device (2) to allow liquid to flow through the flow channel (25) of the microfluidic device (2); a sample liquid source which can be connected to the microfluidic device (2) in order to supply the microfluidic device (2) with a sample liquid (27); a primary separation fluid source connectable to the microfluidic device (2) for supplying the microfluidic device (2) with initial separation fluid (28) for sealing the sample fluid (27) within the array of reaction regions (26); a secondary separation liquid source (4) which can be connected to the microfluidic device (2) in order to supply the microfluidic device (2) with additional separation liquid (29), and a pump device (31) which is connected to the flow circuit (3) and is suitable for pumping the additional separating liquid (29) through the flow channel (25), wherein the microfluidic system further comprises a control device to control the pumping device (31) to pump the additional separation liquid (29) through the flow channel (25) if necessary in order to flush gas bubbles from the flow channel (25). Microfluidic system (1; 1') according to Protection claim 41 , wherein the sample liquid (27) is an aqueous solution comprising the biological sample and the reagents required for the dPCR assay, and wherein each separation liquid (28, 29) is a liquid that is immiscible with the sample liquid (27). Microfluidic system (1; 1') according to Protection claim 42 , wherein each separation liquid (28, 29) is either oil or a polymer liquid, such as a silicone liquid, or a mixture thereof. Microfluidic system (1; 1') according to Protection claim 43 , wherein the initial separation liquid (28) and the additional separation liquid (29) consist of the same separation liquid material. Microfluidic system (1; 1') according to one of Protection claims 41 until 44 , wherein the primary separating liquid source and the secondary separating liquid source (4) are realized by different separating liquid reservoirs or by a common separating liquid reservoir. Microfluidic system (1; 1') according to Protection claim 45 , whereby the initial separating liquid (28) is an untempered separating liquid. Microfluidic system (1; 1') according to one of Protection claims 41 until 46 , wherein the pump device (31) is controlled so that it pumps a predetermined amount of the additional separation liquid (29) through the flow channel (25). Microfluidic system (1; 1') according to Protection claim 47 , wherein the pump device (31) is controlled in such a way that it pumps the additional separating liquid (29) through the flow channel (25) constantly, intermittently or when gas bubbles are detected in the flow channel (25). Microfluidic system (1; 1') according to one of Protection claims 41 until 48 , wherein the microfluidic system (1; 1') further has a bubble trap (32) connected to the flow circuit (3) for separating gas from the separating liquid (28, 29), the bubble trap (32) being located downstream of the outlet (24). the microfluidic device (2) is arranged. Microfluidic system (1; 1') according to one of Protection claims 41 until 49 , wherein the microfluidic system (1; 1') is used to examine the biological sample provided in the form of the sample liquid (27) to each element of the array of reaction areas (26). Microfluidic system (1; 1') according to one of Protection claims 41 until 50 , wherein the inlet (23) and / or the outlet (24) in the form of a connection port for connecting the microfluidic device (2) to the sample liquid source, for connecting the microfluidic device (2) to the primary separation liquid source and / or for connecting the microfluidic Device (2) is designed with the secondary separation liquid source (4). Microfluidic system (1; 1') according to one of Protection claims 41 until 51 , wherein the microfluidic device (2) is constructed with at least one lower layer (21) and an upper layer (22), wherein either the lower layer (21) or the upper layer (22) contains the array of reaction areas (26), the Provides inlet (23) and outlet (24), and wherein the flow channel (25) is arranged between the lower layer (21) and the upper layer (22) and is in fluid communication with the array of reaction regions (26). Microfluidic system (1; 1') according to Protection claim 52 , where each reaction region is a microwell or a nanowell. Microfluidic system (1; 1') according to Protection claim 53 , wherein the microfluidic device (2) is a microfluidic chip Microfluidic system (1; 1') according to one of Protection claims 41 until 54 , wherein the sample liquid (27) is an aqueous solution containing the biological sample and reagents for the dPCR assay. Microfluidic system (1; 1') according to Protection claim 55 , wherein first the sample liquid (27) is flowed through the flow channel (25) into the arrangement of reaction areas (26), and wherein the initial separation liquid (28) is flowed into the flow channel (25) of the microfluidic device (2) after the Sample liquid (27) was supplied to the arrangement of reaction areas (26). Microfluidic system (1; 1') according to one of Protection claims 41 until 56 , wherein the microfluidic system (1; 1') further comprises a detection means for detecting the presence or generation of gas bubbles in the microfluidic device (2). Microfluidic system (1; 1') according to Protection claim 57 , wherein the detection means is an optical imaging device. Microfluidic system (1; 1') according to Protection claim 58 , where the detection means is an optical camera. Microfluidic system (1; 1') according to one of Protection claims 41 until 59 , wherein the microfluidic system (1; 1') further comprises a thermal holder that houses the microfluidic device (2) to impart a thermocycling temperature profile to the array of reaction regions (26). Microfluidic system (1; 1') according to one of Protection claims 41 until 60 , wherein provision of a thermocycling temperature profile for the arrangement of reaction areas (26) is implemented by means of a heating and/or cooling device for heating and/or cooling the temperature of the separating liquid to a desired thermocycling temperature profile temperature. Microfluidic system (1; 1') according to Protection claim 61 , wherein the secondary separation liquid source (4) comprises a storage container (42) with unheated additional separation liquid (29), a flow heating device (5) being provided downstream of the storage container (42) in order to heat the additional separation liquid (29) if necessary. Microfluidic system (1; 1') according to Protection claim 61 or 62 , wherein the secondary separation liquid source (4) comprises at least one reservoir (41) with heated additional separation liquid (29) and at least one reservoir (42) with non-heated or cooled additional separation liquid (29), the reservoirs (41, 42) being separated by a Valve mechanism (411, 421), such as a respective electronically controlled dispensing valve (411, 421) or a common mixing valve, are separably connected to the flow circuit (3). Microfluidic system (1; 1') according to one of Protection claims 61 until 63 , the additional separating liquid (29) being electrically conductive material, such as. B. graphene material, to heat the additional separation liquid (29) by applying electricity to the additional separation liquid if necessary. Microfluidic system (1; 1') according to one of Protection claims 41 until 64 , wherein the microfluidic system (1; 1') further comprises a pressure chamber which surrounds at least the microfluidic device (2).

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