Microfluidic system for digital polymerase chain reaction of biological samples, and respective methods

Abstract

To provide a microfluidic system and respective method for dPCR of a biological sample exhibiting an improved solution for avoiding and / or removing gas bubbles inside a flow channel while maintaining or even improving thermocycling efficiency of the microfluidic system, in the technical field of dPCR assay.SOLUTION: Provided is a microfluidic system (1) having: at least one microfluidic device (2) an inlet (23), an outlet (24), a flow channel (25), and an array of reaction areas (26); a flow circuit (3); a sample liquid source; a primary sealing liquid source for providing an initial sealing liquid (28) for sealing the sample liquid (27) inside the array; a secondary sealing liquid source (4) for providing the microfluidic device (2) with additional sealing liquid (29); and a pumping means (31) connected to the flow circuit (3) and adapted to pump the additional sealing liquid (29) through the flow channel (25).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Generally, the present invention relates to the technical field of sample analysis such as chemical analysis of chemical reactions or biochemical reactions, and more specifically, to the technical field of high-throughput analysis of biological samples. More particularly, the present invention relates to a microfluidic system for digital polymerase chain reaction (dPCR) of biological samples. Even more particularly, such a system comprises a microfluidic device having a flow channel in fluid communication with an array of reaction regions, also referred to as partitions, each of which is implemented as a reaction chamber or reaction vessel and functions as a reaction site for a chemical or biological reaction of at least one biological sample provided therein, for example in the form of wells or micro-wells, and the system can achieve a desired thermocycling temperature profile with a microfluidic device that is as fast and reliable as possible. Further, the present invention relates to each method for dPCR of biological samples in such a microfluidic system.

Background Art

[0002] In the field of diagnostic techniques for chemical analysis of chemical reactions or biochemical reactions, it is the goal to perform multiple different chemical analyses on one or more test samples on the same - preferably disposable - microfluidic device, thereby providing a method for independently analyzing one or more test samples with multiple different reagents during a single analysis process. Such test samples are typically biological samples, which are obtained from patients by medical personnel in a laboratory analysis, for example to determine the concentration levels of different components within the obtained sample. Thus, the terms "sample" and "biological sample" refer to materials that may potentially contain the target component, and the sample may be derived from any biological source, such as physiological fluids including blood, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, feces, semen, milk, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cultured cells, etc., and the sample may be particularly considered to contain a given antigen or nucleic acid.

[0003] Most of the known chemical, biochemical, and / or biological chemical analyses involve immobilizing a biological sample as described above within a reaction site, performing one or more reactions with the immobilized sample material, and then performing a quantitative and / or qualitative analysis process. Here, for many biological, biochemical, diagnostic, or therapeutic applications, it is essential to be able to accurately determine the amount or concentration of a specific substance or compound, i.e., the target component, within the biological sample. To achieve this objective as accurately as possible, various methods have been developed over the years in this technical field, such as the widely known polymerase chain reaction (PCR) method, which enables the in vitro synthesis of nucleic acids within a biological sample and is a cost-effective method by which DNA segments can be specifically replicated, i.e., small segments of DNA or RNA within the sample can be copied or amplified. The development of such methods for amplifying DNA segments or RNA segments has yielded great benefits in genetic analysis as well as in the diagnosis of many genetic diseases or the detection of virus amounts. Generally, thermal cycling, also commonly referred to as thermocycling, is utilized to provide heating and cooling of the reactants within the sample in a reaction chamber for amplifying such DNA segments or RNA segments, and laboratory equipment including a thermocycler is typically used to achieve an automated procedure for diagnostic chemical analysis based on PCR. During the execution of PCR, a liquid PCR sample has to be repeatedly heated and cooled to various temperature levels and maintained at various temperature plateaus for a certain period of time. As an example, during the course of a typical PCR execution, a specific target nucleic acid is such that the nucleic acids present in the reaction mixture are (a) denatured at a relatively high temperature, for example above 90°C, usually at a denaturation temperature of about 94°C to 95°C, for the separation of double-stranded DNA, and (b) the reaction mixture is

[0004] ​The temperature at which the short oligonucleotide primer binds to the single-stranded target nucleic acid, e.g., for primer binding to a separated DNA strand to provide a template (annealing), is cooled to an annealing temperature of about 52°C to 56°C, and then, (c) the primer is extended / progressed using a polymerase enzyme at an extension temperature of about 72°C so that the original nucleic acid sequence is replicated, e.g., to generate a new DNA strand, and is amplified by repeating a series of steps. By repeating the cycle of denaturation, annealing, and extension, usually about 25 to 30 repetition cycles, the amount of target nucleic acid present in the sample increases exponentially. Currently, in order to accurately maintain such a temperature plateau during thermocycling, a uniform temperature distribution should be maintained across the reaction zone so that all reaction regions can be uniformly heated and cooled to obtain a uniform sample yield between the reaction regions containing the sample. To perform a regular PCR method, a generally known thermocycling device such as a thermal cycler / thermocycler for amplifying a DNA segment can be used, which is basically composed of a mount for receiving the sample, often called a sample temperature control mount, and a heat pump attached to the mount, and the heat pipe is often provided in the form of a combination of a Peltier element used for dynamic heating and cooling of the mount and thus for dynamically controlling the temperature provided to the sample, and each heat sink thermally coupled to the Peltier element for dissipating heat, e.g., to the ambient environment.

[0005] Generally, there is a fundamental need to continue to perform diagnostic chemical analyses faster, cheaper, and more simply, while enhancing the efficiency of conventional laboratory processes and achieving high precision. One particular example of the method mentioned for amplifying a DNA segment or an RNA segment, which is the focus of the present invention, is the digital polymerase chain reaction method, also called digital PCR or dPCR, which represents an improvement in biotechnology over the conventional PCR method as described above and can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA, or RNA. Here, while conventional PCR performs one reaction per sample, dPCR performs one reaction within a sample divided into a large number of partitions each provided in a large number of reaction regions, and the reaction is performed individually in each reaction region so as to enable more reliable nucleic acid collection and highly sensitive measurement. Thus, the substantial difference between dPCR and conventional PCR lies in the method of measuring the amount of nucleic acid. Therefore, considerable efforts have been made to increase the number of parallel chemical analyses on a single carrier device and to achieve miniaturization and integration of various chemical analysis operations. As an example of such a single carrier device, microfluidic devices such as microfluidic chips have been developed, which provide microscale channels and microscale reaction regions for receiving samples on the micro-liter or nano-liter scale in the form of a flowable sample liquid such as an aqueous sample liquid. Micro-liter scale reagents, typically pre-filled in an array of small wells such as microwells or nanowells provided as reaction regions on the microfluidic chip, are placed there to contact the flow of the sample liquid flowing through the flow path, and each type of chemical analysis depends on the reagents loaded in the array of reaction regions as well as the configuration of the flow path and the detector. Filling the microfluidic chip with the sample liquid can be carried out by pipetting the sample liquid into the chip. With this advanced technology, multiple chemical analyses can be simultaneously performed on a miniaturized scale. Most of these chemical, biochemical, and / or biological chemical analyses are directed to the immobilization of biological materials such as polypeptides and nucleic acids, cells or tissues in wells, and the performance of one or more reactions with the immobilized materials following quantitative and / or qualitative analysis processes such as luminescence test measurements.For illustration, FIG. 3A schematically shows a top view of an example of a known microfluidic chip 6, and FIG. 3B shows the chip 6 of FIG. 3A in a cross-sectional view taken along line A-A of FIG. 3A. The microfluidic chip 6 consists of a lower plate 61 and an upper plate 62, i.e., a so-called two-layer microfluidic chip. The upper plate 62 provides an inlet 63 for introducing liquid into the chip and an outlet 64 for the liquid to flow out of the chip. The combination of the lower plate 61, i.e., the lower layer of the chip, and the upper plate 62, i.e., the upper layer of the chip, establishes a flow path 65 therebetween. The flow path 65 connects the inlet 63 to the outlet 64. An array of microwells 66 is provided in the flow path 65 on its upper side, i.e., inside the upper plate 62. Here, when the sample liquid is filled into the flow path 65 through the inlet 63 and directed towards the outlet 63, if the sample liquid is made to flow along the array of microwells, the sample liquid will fill each part of the array of microwells 66.

[0006] However, when the volume of the reaction chamber is actually reduced to generate the desired small dimensions, resulting in a microfluidic structure of the microfluidic device, several known problems increase, such as an increase in the surface area-to-volume ratio, or unwanted vaporization of the sample liquid, and in particular, problems associated with the unwanted generation of air bubbles in the liquid provided within or flowing through the microfluidic device. In the focus of the present invention, air bubbles circulating in the microfluidic system can, on the one hand, not only damage the microfluidic structure of any kind of sensor used therein, but also cause unwanted mixing of the samples in adjacent micro-wells, leading to cross-contamination and thus substantial experimental errors and false chemical analysis results, and can therefore mainly damage the biological sample of interest. Thus, air bubbles present in the liquid within the microfluidic structure can pose a serious problem. For example, air bubbles can cause experimental errors in a chromatography column by drying the reaction components within the reaction region. Also, air bubbles can seriously affect the optical detection of the reaction region and the reactions occurring therein, potentially leading to the failure of chemical analysis. In particular, when performing dPCR of a sample in a microfluidic device, for several reasons: (a) air bubbles can be trapped in the microfluidic device when filling it with the sample liquid and the separation liquid, (b) the initially trapped air bubbles (see (a)) can grow due to the evaporation of the sample liquid during thermocycling, or (c) new air bubbles can be generated due to the evaporation of the sample, air bubbles such as air bubbles tend to occur frequently.

[0007] For the exemplary reasons regarding the generation and growth of the bubbles described above, FIGS. 4A-4D schematically show a cross-section of the microfluidic chip 7, which is inserted into respective thermocycling appliances and is shown only schematically. The microfluidic chip 7 has a structure similar to the microfluidic chip 6 of FIGS. 3A and 3B, that is, it includes a lower plate 71 and an upper plate 72, provides an inlet 73, an outlet 74, and a flow path 75 connecting the inlet 73 to the outlet 74, and an array of reaction regions 76 in the form of microwells is provided in the upper side, that is, inside the upper plate 72, in the flow path 75. Here, the array of reaction regions 76 is already filled with the sample liquid 77, and the sealing liquid 78 is introduced into the flow path 75 to seal the sample liquid 77 inside the reaction regions and fill the flow path 75 from the inlet 73 to the outlet 74. To achieve thermocycling of the microfluidic chip 7, the chip 7 is placed inside a thermocycler or a thermal cycler equipped with a bottom heater 81 and a top heater 82 implemented by a thermocycling appliance in the form of a so-called plate cycler 8, that is, a heatable plate also called a hot plate. In FIG. 4A, the microfluidic chip 7 is shown with the heaters 81, 82 in a non-heated state, and air bubbles 91, for example, are erroneously introduced into the flow path 75 directly below the array of reaction regions 76 while filling the microfluidic device 7 with the sample liquid 77 and / or the separation liquid 78.

[0008] Therefore, during filling, the "initial" bubbles 91 may be trapped inside the chip 7, for example, in the sample liquid within the microwells, or in the sealing liquid provided within the flow channels of the chip 7. As seen in FIG. 4B where at least the bottom heater 81 is turned on to provide a heating temperature above 50°C, the already introduced bubbles 91 are growing due to the vaporization of the sample liquid 77 within the array of reaction regions 76. Also, new bubbles 92 are generated due to the unwanted vaporization of the sample liquid 77 within the array of reaction regions 76. Here, the new bubbles 92 may appear and grow within any of the reaction regions during temperature regulation and leave the same in the flow channel 75. In FIG. 4C, the progression of the growth of bubbles 91, 92 due to the unwanted vaporization of the sample liquid 77 is seen, and the flow channel 75 shows bubble growth along its extent, with the growing bubbles 91, 92 being sealed Liquid 78 is gradually extruded from channel 75 and thus exits from inlet 73 and outlet 74, respectively, as indicated by the arrows here. Thereby, growing bubbles 91, 92 remove the sealing liquid 78 from the microwells, and as a result, the sample liquid 77 in the microwells can evaporate more easily. Also, the growing bubbles 91, 92 can spread across multiple microwells, i.e., the bubbles 91, 92 can push aside the sealing liquid 78 from some microwells and "expose" these microwells completely, and as a result, the content of one microwell can move to adjacent microwells. As the heating by the thermal cycler further progresses, the bubbles 91, 92 can grow further and can also fuse into one large bubble 93 (see Fig. 4D), and as a result, the fused and still growing bubble 93 gradually extrudes the sealing liquid 78 further from channel 75 and thus further extrudes from inlet 73 and outlet 74 as indicated by the respective arrows. By the development as shown from Fig. 4A to Fig. 4D, especially Fig. 4D, it can be clearly inferred that due to the generation and growth of unwanted bubbles, the sealing liquid 78 is almost completely extruded from the array of reaction regions 76, thereby invalidating the sealing effect brought about by the sealing liquid 78. Therefore, the reaction components of the sample liquid 77 provided in the array of reaction regions 76 are no longer preserved by the sealing liquid 78 and can thus dry out. In other words, Figs. 4A to 4D show that during thermocycling, the "initial" bubbles 91 and newly emerging bubbles 92 can grow due to the vaporization of the sample liquid 77 provided in the reaction region 76, and the growth of the bubbles continues until the measurement region provided in the form of channel 75 and / or the array of reaction regions 76 is almost empty. Therefore, the removal of the sealing liquid 78 by the bubbles 91, 92, 93 may cause cross-contamination between adjacent reaction regions. Also, since the cell membrane stretches under the force of the unwanted liquid-air interface generated in this way, the bubbles 91, 92, 93 may increase the shear stress on the biological sample material in the reaction region 76.Furthermore, as already mentioned, bubbles can pose a substantial problem for the optical detection of the reaction area and the reactions occurring therein, leading substantially to the failure of chemical analysis that needs to be avoided by all means. Therefore, the removal or avoidance of bubbles is a major challenge in this technical field.

[0009] So far, there have been various approaches to solving the problem of bubbles in the flow channels of microfluidic devices. For example, as described in EP2830769A1, one solution to the problem of bubbles is to provide a specific microfluidic structure from the inlet, such as providing a slit formed along the entire edge of the substrate, so as to prevent bubbles from being introduced into the microfluidic device during filling. The fluid passes through the slit from the inlet manifold and flows into the reaction chamber along almost the entire edge of the substrate at a uniform pressure without bubbles. However, here, the substantial drawback of such a solution is that if such a microfluidic structure cannot prevent bubbles from entering the microfluidic device, the bubbles will reach the reaction chamber and can grow due to the vaporization of the sample at an increased cycle temperature without means to remove the entrant bubbles. Also, the vaporization of the sample at an increased cycle temperature can generate new bubbles, causing the failure of the dPCR method. Therefore, the known solutions provided, even if somewhat effective in avoiding the entry of already existing bubbles into the microfluidic device, still cannot process the bubbles that have entered or newly generated bubbles during thermocycling.

[0010] Another solution to the problem of bubbles is known, for example, from US2005 / 0009101A1, which describes a microfluidic cassette or device that can be used to perform a number of operations on a sample to ultimately result in the detection or quantification of a target analyte, and provides a light pipe that enables the detection of bubbles formed in a hybridization solution or a wash buffer, and provides a roller in a functional relationship with a flexible layer in the sense of a peristaltic interaction to remove the detected bubbles, and the elastomeric material of the flexible layer is used as a peristaltic actuating material. Thus, the solution provided aims to squeeze out any kind of bubbles from the microfluidic device, but this requires an elastomeric fluid chip material. However, the handling and filling of such flexible chip materials are not effective and inaccurate, and the surface modification and optical quality of such materials are low compared to the commonly used non-flexible chip materials, so such a solution is considered not suitable for dPCR chips.

[0011] As a further solution to the problem of bubbles in a microfluidic device caused by the vaporization of a sample liquid, as described, for example, in US2005 / 0148066A1 a pressure can be applied to the microfluidic device, and a device for performing a plurality of simultaneous microchemical and biochemical reactions in an array format is disclosed. Here, the device is thermocycled with a thermal cycler having grooves across its surface, a thin microhole chip is inserted into the grooves and thermocycled, and a thin thermally conductive silicon pad can be used to provide pressure and thermal contact to the surface of the microhole chip to prevent evaporation of the sample in the array and prevent the generation or growth of bubbles. Here, the substantial drawbacks of such a solution are that the overall structure of the device becomes more complex and the additional structure required by applying pressure to the chip is complex.

[0012] Therefore, in the technical field of dPCR chemical analysis, an improved method for avoiding and / or removing bubbles in a flow channel while maintaining or improving the thermal cycling efficiency of a microfluidic system There is a general need to provide a microfluidic system and a respective dPCR method for biological samples that show a solution.

Summary of the Invention

[0013] The inventors of the present invention have confirmed that bubbles can cause serious problems regarding the successful completion of sample thermocycling and can thus lead to substantial failure of chemical analysis. It has also been found that previously proposed solutions are not completely sufficient or satisfactory regarding the avoidance of such bubbles. In particular, the solutions already proposed in the prior art (see above) were either too costly or insufficient to produce reproducible test results. Therefore, the introduction of bubbles into the flow channels of the microfluidic device and the generation and growth of new bubbles during regulation must be avoided, and a newly improved solution was needed. Therefore, due to the fact that when the sealing liquid is flowed through the flow channel, the sealing liquid can reliably wash away the bubbles from the outlet port of the microfluidic chip through the flow channel, the solution of the new invention is basically based on the idea of removing existing or emerging bubbles by "flushing" the microfluidic chip with a separation fluid, namely the sealing liquid, and was developed by the inventors. Therefore, the present invention addresses the above-mentioned problems of a simplified and more effective avoidance and / or removal of bubbles in the flow channels of the microfluidic device within the microfluidic system and at the same time improves its thermocycling efficiency.

[0014] According to a first aspect of the present invention, a microfluidic system for dPCR of a biological sample is provided, which is used for chemically analyzing a biological sample provided to individual reaction regions of an array of reaction regions in the form of a sample liquid, such as an aqueous solution of a polar sample. The microfluidic system has at least one microfluidic device having an inlet, an outlet, a flow path connecting the inlet to the outlet, and an array of reaction regions in fluid communication with the flow path. Here, the microfluidic device can exhibit a structure consisting of at least an upper layer and a lower layer, and either the upper layer or the lower layer can provide an array of reaction regions, an inlet, and an outlet. The flow path is established between the upper layer and the lower layer and is in fluid connection with an array of reaction regions that can be implemented in the form of micro-wells or nano-wells, thereby making the microfluidic device, for example, a microfluidic chip. For example, the overall width of the flow path, also called the lane width, can be in the range of 6 mm to 7 mm, such as 6.4 mm, thereby usually providing a space with a width of about 60 to 100 wells adjacent to each other, that is, in the lateral direction of the flow path. Further, the cross-sectional area of the opening of each well can have a polygon such as a circle, an ellipse, or a hexagon. Due to the polygon of the well opening, especially the hexagonal shape of the well opening, the well openings can be arranged relative to each other at a shorter distance, that is, the distribution density of the well openings in the flow path can be increased. Therefore, the number of wells in the array of wells on the plate can be further maximized. Further, the width of the well opening including the intermediate space between the wells can be 60 μm ≤ w ≤ 110 μm, for example, 62 μm (small well) ≤ w ≤ 104 μm (large well). Further, the microfluidic system has a flow circuit connectable to the microfluidic device for flowing a liquid through the flow path of the microfluidic device, and a sample liquid source connectable to the microfluidic device for supplying a sample liquid to the microfluidic device, for example, by the flow circuit. As the material of the microfluidic device, that is, the device layer, materials such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP) can be used, and the use of COP is preferable, for example, for cost consideration.Furthermore, the flow circuit can be implemented by a tube system, for example, a flexible tube system consisting of one or more flexible tubes. The tube can be composed of, for example, an inner layer of ethylene propylene diene monomer (EPDM) rubber and an outer layer of nitrile butadiene (NBR) rubber potentially reinforced with a synthetic mesh, or generally can be formed from EPDM, NBR, fluorinated ethylene propylene polymer (FEP), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyethersulfone (PES), fluoroelastomer (FKM), silicon, and can furthermore be coated with a heat insulating material.

[0015] Furthermore, the microfluidic system of the present invention includes a primary sealing liquid source connectable to the microfluidic device that provides an initial or primary sealing liquid for sealing the sample liquid in the array of reaction regions to the microfluidic device. The initial sealing liquid can be an unregulated sealing liquid, that is, a non-heated sealing liquid provided, for example, at or near ambient temperature. Furthermore, the microfluidic system of the present invention has a secondary sealing liquid source connectable to the microfluidic device for providing additional sealing liquid to the microfluidic device, and pump means connected to the flow circuit and configured to send out the additional sealing liquid through the flow path. As an example, the pump means of the present invention is one of a peristaltic pump, a metering pump, or a syringe pump, or alternatively, can be provided with additional fluid components such as valves. The pump means of the present invention can be any other type of pump, for example, a diaphragm pump, a wobble piston pump, a micro gear pump, etc. 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 outlet of the microfluidic device can each be implemented in the form of a single connection port, for example, a circular fluid sealing port in the form of a luer lock adapter or the like, for connecting the microfluidic device to a sample liquid source, for connecting the microfluidic device to a primary sealing liquid source, or for connecting the microfluidic device to a secondary sealing liquid source.

[0016] With the above combination of the sealing liquid source and the pumping means of the microfluidic system of the present invention, and the possibility of delivering additional sealing liquid through the flow path, bubbles already present in the flow path, or any type of bubbles that appear during the thermocycling of the sample inside the microfluidic device, can be removed from the microfluidic device by flushing the flow path with the additional sealing liquid. Thus, according to the present invention, the adverse effect of bubbles on the test results can be at least reduced or completely avoided, thereby resulting in a significantly improved microfluidic system.

[0017] In other words, the present invention relates to the general technical field of PCR using a well-plate-based thermocycling structure with a main focus on endpoint digital PCR. More specifically, the well plate is provided in the microfluidic chip in the form of a micro-well plate or a nano-well plate characterized by thousands of nano-wells. Here, the microfluidic chip can be disposable and has a flow path in the form of a closed filling channel on the opening side of the well connected to a macroscopic filling inlet port on one side and an overflow outlet port on the other side. Also, the disposable microfluidic chip can include a plurality of arrays of reaction regions with such micro-well structures, and thus can include a plurality of filling channels on the opening side. Usually, the microfluidic chip can be pre-filled with a so-called PCR master mix that is mixed with the target mix in the form of a sample liquid. Thereafter, the flow path on the opening side of the micro-well is filled with a sealing liquid, such as cover oil, to prevent the sample liquid in the micro-well from vaporizing during thermocycling and to prevent the sample from moving from well to well. Here, the oil can be filled into the flow path using, for example, a peristaltic pump that applies a pressure of about 500 mbar. Finally, the microfluidic system of the present invention can be characterized by different sensors that perform filling checks, correct placement checks, and the like.

[0018] According to certain embodiments of the dPCR microfluidic system of the present invention, the initial sealing liquid and the additional sealing liquid are made of the same sealing liquid material, and any sealing liquid used in the microfluidic system of the present invention can be of the same type of sealing liquid. Further, the sealing liquid needs not to be miscible with the sample liquid. As such immiscible liquids, for example, silicone liquids such as polydimethylsiloxane (PDMS) liquid, or oily liquids or polymer liquids such as a mixture of an oily liquid and a polymer liquid can be used. Further, the initial sealing liquid and the additional sealing liquid can be provided by different sealing liquid sources, or by the same sealing liquid source, that is, the primary sealing liquid source and the secondary sealing liquid source can be implemented by different sealing liquid reservoirs, or by a common sealing liquid reservoir. Specifically, when the initial sealing liquid and the additional sealing liquid are made of the same sealing liquid material or type, the primary sealing liquid source and the secondary sealing liquid source can be implemented by one common sealing liquid reservoir. In this case, the terms "primary" and "secondary" simply refer to the functions of the common sealing liquid reservoir. That is, the common sealing liquid reservoir can function as a main sealing liquid source for providing the initial sealing liquid that mainly seals the samples inside the array of reaction regions of the microfluidic device, and can function as a secondary sealing liquid source for providing the additional sealing liquid for flushing out air bubbles from the flow channels of the microfluidic device. Such a configuration can be selected due to the fact that it can reduce the complexity of the structure of the microfluidic system as compared with the provision of two different sealing liquid reservoirs.

[0019] According to a further specific embodiment of the dPCR microfluidic system of the present invention, the pump means is controlled by a control unit and can, if necessary, send additional sealing liquid through the flow path and wash out air bubbles from the flow path. Here, the control unit can be automatically started by the operator manually or based on a feedback signal from an additional system component that detects or monitors the generation of air bubbles in the flow path, for example. Further, the control unit can instruct the pump means to send additional sealing liquid through the flow path based on a predetermined pattern, for example based on a thermocycling process applied to the sample liquid in the reaction region. Here, it can be advantageous for the pump means to be controlled to send a predetermined amount of additional sealing liquid through the flow path, and the pump means can be controlled to send additional sealing liquid through the flow path continuously, intermittently, or when air bubbles in the flow path are detected.

[0020] According to a further specific embodiment of the dPCR microfluidic system of the present invention, the microfluidic system can further comprise a bubble trap connected to the flow circuit to separate air from the sealing liquid, and the bubble trap is arranged downstream of the outlet of the microfluidic device. Thereby, air bubbles washed out from the flow path of the microfluidic device by the flowed additional sealing liquid can be removed from the sealing liquid flowing through the flow circuit. Thus any bubble trap can be arranged in the flow path of the sealing liquid provided by the flow circuit to separate air bubbles from the flowed sealing liquid. Thereby, air bubbles can be immediately removed from the microfluidic system of the present invention without the need to collect the sealing fluid exiting the microfluidic device and extract air bubbles therefrom.

[0021] According to another specific embodiment of the dPCR microfluidic system of the present invention, the sample liquid can be an aqueous solution containing a biological sample and reagents necessary for dPCR chemical analysis. Here, first, the sample liquid can flow into the array of reaction regions through the flow path in order to fill each reaction region with the sample liquid. Then, that is, after providing the sample liquid to the array of reaction regions, an initial sealing liquid flows into the flow path of the microfluidic device in order to seal the sample liquid in the reaction regions.

[0022] According to a further specific embodiment of the dPCR microfluidic system of the present invention, when the flow path allows optical monitoring, the microfluidic system can further include detection means for detecting and / or monitoring the presence or generation of air bubbles in the flow path during or before thermocycling, such as an optical imaging device, for example an optical camera, etc., for detecting the presence or generation of air bubbles in the microfluidic device. Here, for example, the inside of the flow path can be visually recognized from the outside by, for example, an observation window, a transparent wall of the flow path, etc. Therefore, the detection means can be used to provide a feedback signal indicating the generation of air bubbles in the flow path, and the control unit can be started based on such a feedback signal. Therefore, with such a structure, the microfluidic system can operate automatically without the need for an operator to monitor the microfluidic device during thermocycling or the like.

[0023] In order to be able to provide a thermocycling temperature profile to the sample material in the reaction regions, the microfluidic system can include a thermal mount for receiving the microfluidic device for providing a thermocycling temperature profile to the array of reaction regions. For example, the thermal structure described in connection with any one of FIGS. 4A to 4D is used, that is, a so-called plate cycler can accommodate the microfluidic device of the microfluidic system of the present invention. Alternatively, the provision of a thermocycling temperature profile to the array of reaction regions is implemented by heating and / or cooling means for heating and / or cooling the temperature of an additional sealing liquid to the desired thermocycling temperature profile temperature. Thus, the desired thermocycling temperature profile of the contents of the reaction region can be achieved not by applying heat by an external heat source thermally connected to the microfluidic device, but by a heated or cooled sealing liquid flowing through the flow channels of the microfluidic device, i.e., a new method of applying heat to the sample material while being able to wash away any kind of bubbles in the flow channels. Thus, such a solution can achieve not only bubble removal but also heating and / or cooling of biological samples in the reaction region from within the microfluidic device. Thus, a more direct and faster application of the thermocycling temperature profile to the biological sample can also be achieved, and known external components such as a top heater and a bottom heater can be omitted, thus simplifying the known structure of the thermocycling equipment. Here, instead of applying heat from the outside, there are several options that can be combined with each other as needed to achieve such temperature control from within the microfluidic device, and the options may include the following.

[0024] (a) The secondary sealing liquid source includes a reservoir with additional unheated sealing liquid and is provided with flow-type heating means downstream of the reservoir for heating the additional sealing liquid as needed, i.e., for heating the sealing liquid flowing through the flow-type heating means when required by the thermocycling temperature profile. The flow-type heating means can be implemented in the form of temperature control means provided around a part of the flow circuit coming from the sealing liquid reservoir, connected flow heater, etc.; (b) The secondary sealing liquid source includes at least one reservoir with heated additional sealing liquid and at least one reservoir with unheated or cooled additional sealing liquid. The reservoirs can be separably connected to the flow circuit by respective valve mechanisms such as electronically controlled delivery valves, or the reservoirs can be connected to the flow circuit by a mixing valve for mixing the heated additional sealing liquid and the unheated or cooled additional sealing liquid to achieve the desired temperature of the sealing liquid supplied to the microfluidic device; (c) The additional sealing liquid includes a conductive material such as a graphene material for heating the additional sealing liquid by applying power to the additional sealing liquid as needed.

[0025] (a) and (b) When using any one or a combination thereof, the pumping means must be configured to be able to follow the desired dPCR cycle, that is, to place the additional sealing liquid adjusted to the temperature on the sample array respectively, then separate from it again, and send out any additional amount of additional sealing liquid through the flow path to remove air bubbles. When using option (c), that is, when the sealing liquid is electrically heated, the liquid should be a conductive liquid that provides an appropriate resistance so that it is heated while an electric current flows. Here, a graphene solution is suitable for such applications. However, in such cases, the sealing liquid cannot be electrically cooled. Instead, when it is necessary to lower the sample temperature during dPCR, the reservoir or the microfluidic chip itself needs to be cooled. Furthermore, when an electrically heated solution is applied, a circulating sealing liquid system can be used without the need for a reservoir that requires precise control of the dPCR cycle, for example, by an extensive sensor structure that monitors temperature and current in real time.

[0026] According to a further specific embodiment of the dPCR microfluidic system of the present invention, the microfluidic system may further include at least a pressure chamber surrounding the microfluidic device. Here, the function of such an additional pressure chamber is to add to the already achieved avoidance or removal of air bubbles generated within the microfluidic system of the present invention, thereby further ensuring that the air bubbles cannot interfere with the chemical analysis results. Regarding thermocycling itself, a disposable microfluidic device including an oil filling port can be set to a pressure of 1 to 2 bar, for example, about 1.5 bar, in order to further suppress the generation of air bubbles during thermocycling. Here, the pressure is preferably applied inside the microfluidic device by placing the entire microfluidic device within the pressure chamber.

[0027] According to a further specific embodiment of the present invention, the apparatus of the present invention can further comprise at least one sensor for controlling the temperature of a biological sample received in a microfluidic device, and such a sensor can be a temperature sensor, for example, combined with a flow sensor. Thus, by providing respective sensors to any of the components of the microfluidic system of the present invention, the temperature of the biological sample during dPCR can be closely monitored, and the heating / cooling function of the microfluidic system can be controlled based on the measured temperature values of the sample to accurately and efficiently adjust different temperature plateaus of dPCR. Therefore, such sensors, such as temperature sensors, enable accurate control of thermocycling temperatures, such as by a control algorithm, and the temperature sensor and other sensors are used to control their respective heating / cooling rates, and the heating / cooling power can be significantly varied by changing the pump rate of the fluid.

[0028] According to another aspect of the present invention, a method for dPCR of a biological sample in a microfluidic system as described above is provided, the method comprising flowing a sample liquid through a flow channel of a microfluidic device, particularly provided in the form of a microfluidic chip, and continuously filling an array of reaction regions with the sample liquid by pushing the sample liquid through the flow channel, sealing each reaction region after filling the reaction regions with the sample liquid, and pushing the remaining sample liquid out of the microfluid device by flowing an initial sealing liquid into the flow channel of the microfluidic device, applying a thermocycling temperature profile to the array of reaction regions, and flowing an additional sealing liquid into the flow channel of the microfluidic device to wash out air bubbles from the flow channel.

[0029] Here, the step of applying a thermocycling temperature profile to the array of reaction regions and the step of delivering additional sealing liquid to the flow channels of the microfluidic device to wash out air bubbles from the flow channels can be provided during the process of the combined steps, i.e., it can be provided when the application of temperature to the array of reaction regions is achieved by delivering heated additional sealing liquid through the flow channels, and the delivery of the additional sealing liquid heats the sample liquid in the reaction array while washing out air bubbles from the flow channels. Further, preferably, the dPCR method of the present invention also includes the step of chemically analyzing the biological sample provided in the array of reaction regions, thereby making the method an analysis method based on dPCR.

[0030] According to certain further embodiments, the step of delivering additional sealing liquid through the flow channels can include delivering a predetermined amount of additional sealing liquid through the flow channels as needed, and the step of delivering additional sealing liquid through the flow channels can be, for example, the step of continuously delivering additional sealing liquid to the flow channels only when it is necessary to thermoregulate the sample liquid in the reaction region, or the step of intermittently pumping additional sealing liquid into the flow channels, and / or the step of delivering additional sealing liquid through the flow channels when air bubbles in the flow channels are detected. Here, since there is a possibility of delivering additional sealing liquid to the flow channels as needed, air bubbles already present in the flow channels or any type of air bubbles that appear during the thermocycling of the sample in the microfluidic device can be removed from the microfluidic device by washing out the flow channels with the additional sealing liquid. Thus, in the method of the present invention, the adverse effect of air bubbles on the test results can be at least reduced or completely avoided, thereby providing a significantly improved method for dPCR of biological samples in the microfluidic system of the present invention.

[0031] According to certain embodiments of the dPCR method of the present invention, the method can further include monitoring and detecting the presence or generation of bubbles in the microfluidic device by means of, for example, a detection means in the form of an optical camera, etc., when the flow path allows optical monitoring, i.e., when the inside of the flow path can be seen from the outside, for example, by a display window of the flow path, a transparent wall, etc., before and during thermocycling. Thus, the detection means can be used to provide a feedback signal indicating the generation of bubbles in the flow path, and the control unit can be operated based on such a feedback signal, resulting in a feedback-controlled dPCR method. Alternatively or additionally, the method of the present invention can further include separating bubbles from the additional sealing liquid, for example, by a bubble trap connected to the flow circuit downstream of the outlet of the microfluidic device. Thereby, the bubbles flowing out from the flow path of the microfluidic device by the additional sealing liquid flowing through can be removed from the sealing liquid flowing in the flow circuit. Thus, the optional step of separating bubbles from the additional sealing liquid by a bubble trap disposed in the flow path of the sealing liquid can be useful for immediately removing bubbles from the microfluidic system of the present invention without collecting the sealing liquid exiting the microfluidic device and extracting bubbles therefrom at a later stage. Alternatively or additionally, the method of the present invention can also include applying pressure to the microfluidic device, for example, by at least a pressure chamber surrounding the microfluidic device. The function of such an additional pressure application step can be added to the already achieved avoidance or removal ability of the dPCR method of the present invention, thereby further ensuring that bubbles do not interfere with the test results. Thereby, the microfluidic device can be set under a pressure of 1 to 2 bar, for example, about 1.5 bar, to further suppress the generation of bubbles during thermocycling, and pressure can be applied to the inside of the microfluidic device by applying pressure to the side walls of the flow path that function as flexible pressure transmission elements, or in such a case, by applying pressure to the entire microfluidic device that needs to exhibit a specific compression ratio.

[0032] According to certain embodiments of the dPCR method of the present invention, the step of applying a thermocycling temperature profile to an array of reaction regions may include controlling the temperature profile of a thermal mount that receives the microfluidic device to provide the thermocycling temperature profile to the array of reaction regions, or alternatively controlling heating and / or cooling means for heating and / or cooling the sealing liquid temperature to a desired thermocycling temperature profile temperature. Here, the step of controlling the heating and / or cooling means can include any of the following options. (a) Controlling flow-type heating means provided downstream of a secondary sealing liquid source to heat additional sealing liquid as needed. (b) Controlling a valve mechanism for mixing additional sealing liquid from at least one reservoir with heated additional sealing liquid and at least one reservoir with unheated or cooled additional sealing liquid. (c) Applying a voltage to additional sealing liquid as needed, wherein the additional sealing liquid includes a conductive material such as a graphene material for heating the additional sealing liquid by application of power. (d) Any combination of steps (a) through (c) of the foregoing options.

[0033] The above-described microfluidic system of the present invention and the method of each invention can be part of an automated processing system such as an analytical, pre-analytical, or post-analytical processing system. The automated processing system is commonly used in state-of-the-art laboratories for automatically processing biological samples and includes any device or device component operable to perform one or more processing steps / workflow steps on one or more biological samples, covering analytical instruments, pre-analytical instruments, and post-analytical instruments. The expression "processing step" refers to a physically executed processing step, such as performing a specific step of dPCR implementation. As used herein, the term "analysis" encompasses any processing step performed by one or more experimental devices or operating units operable to perform an analytical test on one or more biological samples. In the context of biomedical research, the analytical process is a technical procedure for characterizing the parameters of a biological sample or analyte. Such characterization of parameters includes, for example, determining the concentrations of specific proteins, nucleic acids, metabolites, ions, or molecules of various sizes in biological samples derived from, for example, humans or experimental animals. The information collected can be used, for example, to evaluate the effects of drug administration on an organism or a specific tissue. Further analysis can determine optical, electrochemical, or other parameters of the biological sample or analyte contained in the sample substance.

[0034] The above method steps can be controlled by the control unit of the above microfluidic system, which can also control any kind of operation, monitoring or control of the above microfluidic system and its components. As used herein, the term "control unit" includes physical or virtual processing devices such as a CPU, which can also control the entire experimental equipment or the entire workstation including one or more experimental equipment in the way the workflow and workflow steps are executed. The control unit can, for example, be equipped with different types of application software and instruct an automated processing system or a specific device or its apparatus to execute pre-analysis, post-analysis, and analysis workflow steps. The control unit can receive information from the data management unit regarding which steps need to be executed for a specific sample. Further, the control unit may be integrated with the data management unit, may be included in a server computer, and / or may be part of one device or distributed among multiple devices of an automated processing system. The control unit may be embodied, for example, as a programmable logic controller that executes a computer-readable program with instructions for performing operations. Here, a user interface can be further provided to receive such instructions from the user. As used herein, the term "user interface" includes any suitable part of application software and / or hardware for the interaction between the operator and the machine, and includes, but is not limited to, a graphical user interface that receives commands from the operator as input, provides feedback, and conveys information therein. Also, the system / device may expose several user interfaces to provide services to different types of users / operators.

[0035] As also used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. Similarly The words "comprise", "contain", and "encompass" should be construed inclusively rather than exclusively. That is, it means "including but not limited to". Similarly, the word "or" is intended to include "and" unless clearly indicated otherwise in the context. The terms "plurality", "multiple", or "multitude" refer to two or more with an integer multiple, i.e., 2 or >2, and the terms "single" or "sole" refer to one, i.e., =1. Further, the term "at least one" should be understood as one or more, i.e., 1 or >1 with an integer multiple. Therefore, words using singular or plural also include plural and singular respectively. Further, the words "here", "above", "before", and "below", and words of similar import, when used in this application, refer to the whole of this application rather than a particular part of the application.

[0036] The description of specific embodiments of the present invention is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but as will be recognized by those skilled in the art, various equivalent modifications are possible within the scope of the disclosure presented by the appended claims. Specific elements of the foregoing and following embodiments can be combined or substituted with elements of other embodiments. Also, in the drawings, the same reference numerals are used to indicate the same elements to avoid repetition, and parts that can be easily implemented by those skilled in the art may be omitted. Further, although the advantages associated with specific embodiments of the present disclosure are described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit advantages such that they are included within the scope of the disclosure defined by the appended claims.

[0037] The following examples are intended to illustrate specific embodiments of the present invention. Accordingly, the specific implementations described below should not be construed as limitations on the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention as defined by the appended claims, and accordingly, it should 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 specific embodiments shown in the drawings.

Brief Description of the Drawings

[0038]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Best Mode for Carrying Out the Invention

[0039] Figure 1 schematically shows a microfluidic system 1 for dPCR of a biological sample according to a first embodiment of the present invention, with the main components provided as an exemplary cross-sectional view or emoji. The microfluidic system 1 shown in Figure 1 includes a microfluidic device 2 in the form of a microfluidic chip, and the device 2 shows a structure similar to the state-of-the-art chip 7 shown in Figure 3B. The device 2 substantially consists of a lower plate 21 and an upper plate 22, and provides an inlet 23, an outlet 24, and a flow path 25 connecting the inlet 23 to the outlet 24. Further, an array of reaction regions 26 in the form of micro-wells or nano-wells is provided inside the upper plate 22, i.e., on the upper side of the flow path 25, so as to enable monitoring of the reactions in the reaction regions 26 from above. Further, in the illustrated state, the microfluidic device 2 is already flushed with a sample liquid 27 that fills the array of reaction regions 26, i.e., each single micro-well of the array of reaction regions 26. Such filling of the sample liquid 27 into the array of reaction regions 26 can be performed at a filling station (not shown) for pre-placing the microfluidic device 2 within the microfluidic system 1, and the filling station (not shown) is considered as the part of the microfluidic system 1 used to introduce the sample liquid 27 into the array of reaction regions 26. Also, an initial sealing liquid 28 is already filled in the microfluidic device 2, and filling the flow path 25 with the initial sealing liquid 28 to seal the sample liquid 27 within the array of reaction regions 26 and to seal the array of reaction regions 26 can also be performed at a filling station (not shown) before placing the microfluidic device 2 within the microfluidic system 1. Alternatively, filling the flow path 25 with the initial sealing liquid 28 to seal the array of reaction regions 26 may be performed by the microfluidic system 1 itself with an additional sealing liquid 29, and the part of the additional sealing liquid 29 that actually seals the sample liquid 29 within the array of reaction regions 26 should be understood as the "initial" sealing liquid 28.Here, the filling station is connectable to the microfluidic device 2 to provide the sample liquid 27 to the microfluidic device 2, and the filling station (not shown) can be considered as a connectable combined functional part of the microfluidic system 1, which is a sample liquid source and a primary sealing liquid source.

[0040] The microfluidic system 1 further comprises a flow circuit 3 connected to the microfluidic device 2. The flow circuit 3 is used to flow the additional sealing liquid 29 through the flow channel 25 of the microfluidic device 2, mainly for flowing the additional sealing liquid 29 through the flow channel 25 of the microfluidic device 2. Here, the flow of the additional sealing liquid 29 is indicated by a circle with a counterclockwise arrow. The additional sealing liquid 29 substantially originates from a secondary sealing liquid source 4 consisting of a temperature-controlled reservoir 41 containing the heated additional sealing liquid 29 and a non-temperature-controlled reservoir 42 containing the unheated or cooled additional sealing liquid 29. The reservoirs 41 and 42 are connected to the flow circuit 3 by electronic control delivery valves 411 and 421 respectively. Therefore, by controlling the delivery valve 411, the heated additional sealing liquid 29 can be introduced into the flow circuit 3, and by controlling the delivery valve 421, the unheated additional sealing liquid 29 can be introduced into the flow circuit 3, thereby obtaining the desired temperature profile of the additional sealing liquid 29 in the flow circuit 3. Alternatively, the temperature-controlled reservoir 41 and the non-temperature-controlled reservoir 42 share a common mixing valve. The common mixing valve is connected to the flow circuit 3 and functions as a mixing faucet, and the pre-mixed and temperature-controlled additional sealing liquid 29 is introduced into the flow circuit 3 and is ready to be provided to the microfluidic device 2.

[0041] Here, to provide additional sealing liquid 29 to flow path 25, a peristaltic pump 31 that functions as pump means of the microfluidic system 1 is provided as part of the flow circuit 3 to send the additional sealing liquid 29 to the microfluidic device 2 so as to exit from its inlet 23, through the flow path 25 and out of the outlet 24. Thereby, not only heat or cold air is provided to the array of reaction regions 26 for thermocycling of samples within the array of reaction regions 26, but also any bubbles that occur inside the flow path 25 are flushed out from the flow path 25 and the outlet 24, that is, from the microfluidic device 2. Here, the pump 31 can continuously extrude the additional sealing liquid 29 through the flow path 25, or can intermittently send the additional sealing liquid 29 to the flow path 25 only when it is necessary to thermoregulate the sample liquid 27 within the array of reaction regions 26, for example. Therefore, due to the possibility of sending the additional sealing liquid 29 to the flow path 25 as needed, existing bubbles within the flow path 25 or any type of bubbles that appear during thermocycling of the sample liquid 27 can be removed from the microfluidic device 2 by flushing the flow path 25 with the additional sealing liquid 29. To enable the ultimate removal of bubbles from the entire flow system, the microfluidic system 1 further includes a bubble trap 32 disposed downstream of the outlet 24 of the microfluidic device 2 and, in this embodiment, in front of the pump 31. The bubble trap 32 is used to separate bubbles from the flowing additional sealing liquid 29. Thereby, the bubbles can be removed immediately from the microfluidic system 1 if any. Therefore, the adverse effect of bubbles on the test results can be at least reduced or completely avoided.

[0042] Figure 2 shows a second embodiment of the microfluidic device 1' of the present invention, which basically has the same structure as that described with respect to the first embodiment shown in Figure 1. Here, the same reference numerals indicate the same elements, and each description is omitted to avoid duplication. However, in contrast to the first embodiment, the microfluidic device 1' includes only a non-thermostatted reservoir 42 containing an additional non-heated sealing liquid 29, and a flow heater 5 is provided downstream of the reservoir 42 to thermostatically control the non-thermostatted reservoir 42 according to the desired thermocycling temperature profile applied to the samples in the array of reaction regions 26. Here also, the reservoir 42 is connected to the flow circuit 3 by respective electronically controlled delivery valves 421, and by controlling the delivery valves 421, not only provides the desired thermocycling temperature profile to the materials in the array of reaction regions 26, but most importantly, an additional sealing liquid 29, each temperature-controlled, can be introduced into the flow circuit 3 and pumped out of the microfluidic device 2 by the pump 31 to wash away the bubbles generated in the flow channel 25.

[0043] According to another embodiment, the structure of the foregoing embodiment shown in FIG. 2 without the flow heater 5 can be applied to the plate cycler 8 known in the art and described in connection with any of FIGS. 4A-4D, and the plate cycler 8 is provided with the microfluidic device 2. Thereby, the provision of the desired thermocycling temperature profile to the sample liquid 27 in the array of reaction regions 26 can be established by the plate cycler 8, and the delivery of the additional sealing liquid 29 that is not heated is only used for removing air bubbles, if present, in the flow path 25. Further, according to another alternative embodiment, the structure of the embodiment shown in FIG. 2 is applicable without the flow heater 5 or the plate cycler 8, but the additional sealing liquid 29 includes a conductive graphene material for heating the additional sealing liquid 29 by applying a voltage to the additional sealing liquid 29 as needed, thereby achieving heating of the additional sealing liquid 29 so that a desired thermocycling temperature profile can be applied to the sample material in the array of reaction regions 26 without the need for any kind of external heater or heating reservoir.

[0044] Although the present invention has been described in connection with its specific embodiments, it should be understood that this description is for illustrative purposes only. Accordingly, the present invention is intended to be limited only by the appended claims.

Description of Reference Numerals

[0045] 1 Microfluidic system (first embodiment) 1´ Microfluidic system (second embodiment) 2 Microfluidic device 21 Lower plate / lower layer of microfluidic device 22 Upper plate / upper layer of microfluidic device 23 Microfluidic device inlet 231 Microfluidic device inlet cover 24 Microfluidic device outlet 241 Microfluidic device outlet cover 25 Microfluidic device flow path 26 Reaction region / well array 27 Sample liquid (filled in the reaction region) 28 Initial sealing liquid 29 Additional sealing liquid 3 Flow circuit 31 Pump 32 Bubble trap 4 Secondary sealing liquid source 41 Temperature-controlled reservoir 411 Electronically controlled delivery valve 42 Uncontrolled temperature reservoir 421 Electronically controlled delivery valve 5 Flow heater 6 Microfluidic chip 61 Lower plate / layer of the microfluidic chip 62 Upper plate / layer of the microfluidic chip 63 Microfluidic chip inlet 64 Microfluidic chip outlet 65 Microfluidic chip flow path 66 Array of microwells 7 Microfluidic chip 71 Lower plate / layer of the microfluidic chip 72 Upper plate / layer of the microfluidic chip 73 Microfluidic chip inlet 74 Microfluidic chip outlet 75 Microfluidic chip flow path 76 Reaction region / well array 77 Sample liquid (filled in the reaction region) 78 Sealing liquid 8 Plate cycler 81 Bottom heater of the plate cycler 82 Top heater of the plate cycler 91 Initial bubbles 92 Newly emerged bubbles 93 Merged bubbles

Claims

Claim 1 A microfluidic system (1; 1') for digital polymerase chain reaction dPCR of a biological sample, comprising: At least one microfluidic device (2) having an inlet (23), an outlet (24), a flow channel (25) connecting the inlet (23) to the outlet (24), and an array of reaction regions (26) in fluid communication with the flow channel (25); A flow circuit (3) connectable to the microfluidic device (2) for flowing a liquid through the flow channel (25) of the microfluidic device (2); A sample liquid source connectable to the microfluidic device (2) for providing a sample liquid (27) to the microfluidic device (2); A primary sealing liquid source connectable to the microfluidic device (2) for providing an initial sealing liquid (28) to the microfluidic device (2) to seal the sample liquid (27) inside the array of reaction regions (26); A secondary sealing liquid source (4) connectable to the microfluidic device (2) for providing an additional sealing liquid (29) to the microfluidic device (2); Pump means (31) connected to the flow circuit (3) and configured to pump the additional sealing liquid (29) through the flow channel (25); and The pump means (31) is controlled to pump the additional sealing liquid (29) continuously, intermittently, or upon detection of air bubbles in the flow channel (25) through the flow channel (25).

Images