Liquid dispensing system, microfluidic sample carrier sealing system, and method of dispensing a sealing liquid using the same

Abstract

This invention relates to a dispensing system for supplying a pre-defined small volume of liquid (≤200 μl) to a filling region of a microfluidic sample holder, the microfluidic sample holder including at least one flow channel. The system particularly includes at least one interface unit providing for each flow channel of the microfluidic sample holder, wherein each interface unit includes an injector having an injection channel, wherein the cross-section of the outlet portion of the injection channel is larger than the cross-section of the inlet portion and the cross-section of the middle portion of the injection channel. Furthermore, this invention relates to a corresponding microfluidic sample holder sealing system and a corresponding method for dispensing a sealing fluid into the microfluidic sample holder.

Description

Technical Field

[0001] This invention relates generally to the field of sample analysis preparation technology, such as biological sample analysis, and further to the field of preparation technology for high-throughput analysis of biological samples.

[0002] In particular, the present invention relates to a dispensing system for providing a predetermined liquid volume to a filling region of a microfluidic sample holder, the microfluidic sample holder including at least one flow channel providing a micropore array as a reaction chamber for chemical or biological reactions. Here, the objective is often to enable multiple different determinations of one or more test samples on the same (typically disposable) microfluidic sample holder via, for example, thermal cycling. Therefore, in order to further process the test samples, the micropore array pre-filled with the test samples and, for example, multiple different reagents must be sealed to avoid cross-contamination. Thus, in a single analysis, only a small amount of test sample is needed to independently analyze one or more test samples using multiple different reagents. Based on this, the present invention also relates to a microfluidic sample holder sealing system that uses the aforementioned liquid dispensing system to fill such a microfluidic sample holder with a predetermined small amount of sealing liquid, and also relates to a method for dispensing the sealing liquid into the pre-filled microfluidic sample holder by means of the aforementioned microfluidic sample holder sealing system. In other words, the present invention relates to an improved system and method by which a sealing fluid can be used as fully and efficiently as possible in a microfluidic sample holder. Background Technology

[0003] In the field of diagnostic technology for chemical or biochemical reaction assays, a primary objective is to enable the performance of multiple different assays on one or more test samples on the same (preferably disposable) microfluidic sample holder, thereby providing a method for independently analyzing one or more test samples using multiple different reagents in a single analysis. To accurately achieve this objective, various methods have been developed in recent years, such as the well-known polymerase chain reaction (PCR), which, for example in the form of real-time PCR, digital PCR (dPCR), or multiplex PCR, can synthesize nucleic acids in biological samples in vitro, thereby specifically replicating DNA fragments. This method is a cost-effective way to replicate or amplify small fragments of DNA or RNA in a sample.

[0004] Clearly, there is a desire to make diagnostic assays such as dPCR faster, cheaper, and easier to perform, while maintaining the accuracy and efficiency of routine laboratory procedures. Significant efforts have been made in this regard to improve the miniaturization and integration of various assay operations, enabling an increase in the number of parallel assays performed on a single microfluidic sample holder. As an example of such microfluidic sample holders, microfluidic devices such as microfluidic chips (also known as digital polymerase chain reaction (dPCR) chips) have been developed, providing microchannels and microreaction regions for receiving microliters or nanoliters of flowable sample liquids (such as aqueous sample liquids). Microliters of reagent (typically pre-filled into a series of wells, i.e., micropores or nanopores on the microfluidic chip that serve as reaction regions) are added to contact the sample liquid flow through the flow channels. The various assay types depend on the reagents loaded into the array of reaction regions and the configuration of the flow channels and detectors, where sample liquid filling into the microfluidic chip can be achieved by transferring the sample liquid into the chip. This advanced technology allows for the simultaneous performance of multiple assays at a miniaturized scale. Most of these chemical, biochemical, and / or biological assays involve immobilizing biological materials such as peptides and nucleic acids, cells, or tissues in wells, reacting the immobilized material with one or more reactions, and then performing quantitative and / or qualitative analytical processes such as luminescent detection measurements.

[0005] Typically, to perform dPCR assays, a known dPCR chip is first filled with an aqueous dPCR reaction mixture, which usually consists of a biological sample and a PCR master mixture. The dPCR reaction mixture is introduced into the inlet opening using a pipette or similar device and typically flows passively into the well array of the chip by capillary force until the capillary filling process stops. Subsequently, an immiscible separation or sealing solution, such as silicone oil, is forced through the inlet opening into the flow channel. This channel first pushes any remaining dPCR reaction mixture into any remaining empty wells and covers the filled wells, thereby fluidly isolating each well from its surroundings, and especially from each other, to avoid any sample condensation, cross-contamination, or contamination. After the initial filling and subsequent sealing processes are completed, the dPCR chip is typically thermally cycled. In a typical PCR implementation, a specific target nucleic acid is amplified by repeating a series of steps in which the nucleic acids present in the dPCR reaction mixture are (a) denatured at relatively high temperatures (e.g., denaturation temperatures above 90°C and typically 94-95°C) to separate double-stranded DNA; then (b) the reaction mixture is cooled to the temperature at which short oligonucleotide primers bind to single-stranded target nucleic acids (e.g., an annealing temperature of approximately 52-56°C), allowing the primers to bind to the separated DNA strands to provide a template (annealing); and then (c) the primers are extended / extended using polymerase, for example, at an extension temperature of approximately 72°C, to form new DNA strands, allowing the original nucleic acid sequence to be replicated. Typically, each well containing one or more targets will generate a positive signal, where, after thermal cycling, the ratio of positive to negative signals will allow for accurate calculation of the initial target concentration in the sample, for example, by means of luminescent detection measurements. This type of technique allows for the simultaneous execution of multiple assays on a miniaturized scale.

[0006] However, when minimizing the volume of the reaction chamber to create a microfluidic sample holder such as the aforementioned dPCR chip microfluidic structure, several known problems arise to achieve the desired small size, such as the undesirable introduction of air bubbles into the microfluidic sample holder. As the focus of this study, air bubbles present within the microfluidic structure of the microfluidic sample holder can pose a serious problem. Air bubbles circulating through such microfluidic systems not only damage the microfluidic structure of any type of sensor used therein, but also spoil the biological sample of interest by causing undesirable sample mixing in adjacent wells, leading to unwanted cross-contamination and thus significant experimental errors and erroneous measurement results. Therefore, air bubbles trapped within the microfluidic sample holder not only distort the detection signal, but also, when using such a microfluidic sample holder for dPCR, even thermal expansion to the maximum required thermal cycling temperature of approximately 95°C makes it impossible to ensure safe separation of adjacent wells and highly likely to cause undesirable cross-contamination.

[0007] Therefore, it is necessary to achieve the filling of microfluidic sample holders with sealing fluids to avoid contamination and accidental introduction of air bubbles, thereby preventing initial air bubble trapping within the orifices. In this regard, the clean and controlled droplet breakage behavior of the sealing fluid at its dispenser is one of the characteristics, or even the most important, of the sealing fluid filling process when filling microfluidic sample holders with it. This behavior is influenced by various physical variables of the sealing fluid itself, such as viscosity, contact angle, and surface tension. Due to gravity, sealing fluids typically exhibit low surface tension, causing them to spread out of the dispenser, thus increasing the risk of droplet breakage. Accordingly, the precise delivery of small volumes, such as approximately ≤ 200 µl, becomes very challenging due to the rather uncertain droplet breakage behavior caused by the specific properties of the sealing fluid. In addition, the kinetic energy of falling droplets can generate droplets upon impact. These droplets splash uncontrollably and can contaminate the environment. Therefore, during the filling process of microfluidic sample holders, the defined and controlled release of the sealing fluid is crucial to prevent the generation of air bubbles and contamination due to uncontrolled droplet breakage or droplet splashing during dispensing. In this regard, it is important to consider that silicone oils have a much higher viscosity and simultaneously a reduced contact angle and surface tension. This means that such sealants tend to spread wetting, implying that they do not form droplets on the surface or only form droplets for a short period, thus distributing throughout the perimeter of the dispensing point depending on surface conditions, surface energy, surface tension, and contact angle. For example, due to the low surface tension, the hydrostatic effects on the sealant must be considered, as seals, such as those for water sealing, may be prone to failure due to the sealant, leading to spreading leaks. This behavior can result in cross-contamination between two sample holders and, over time, compromise the functionality of peripheral equipment.

[0008] Various solutions have been proposed in the past to overcome the aforementioned problems and control the problematic behavior of the sealing fluid. However, previous solutions have not provided satisfactory results because they have not adequately considered hydrostatic effects, material properties, or rinsing behavior. Therefore, this invention focuses on the requirement that the sealing fluid first flows into the appropriate dispensing system and then is precisely discharged into the microfluidic sample holder without contaminating the surrounding area or introducing unwanted air bubbles. Summary of the Invention

[0009] The present invention addresses the aforementioned problems by means of a dispensing system for supplying a pre-defined small volume of liquid (≤ 200 µl) to a filling region of a microfluidic sample holder, the microfluidic sample holder including at least one flow channel. The dispensing system of the present invention includes a reservoir, such as a sealing fluid reservoir for supplying a high-viscosity sealing fluid, wherein the sealing fluid can exhibit a viscosity up to 100 mm. 2 The kinematic viscosity is 20 mN / m and the surface tension is up to 20 mN / m, such as silicone oil; a liquid pump is connected to the reservoir; and at least one interface unit, such as in the form of a dPCR chip, is provided for each flow channel of a microfluidic sample holder, wherein each interface unit is used to supply liquid to each flow channel. The dispensing system of the present invention further includes: a conduit member including a supply channel connecting the liquid pump and each interface unit; a first valve connected to the supply channel, also referred to as a perfusion valve; and at least one second valve disposed between the supply channel and the corresponding interface unit, also referred to as a switching valve. Here, the first valve of the dispensing system of the present invention may be connected to the downstream end of the supply channel and may be used as a waste valve. The term "downstream" specifies a position in the supply channel of the above-described liquid guiding system that is below the source of the liquid flow, i.e., the reservoir. A similar definition applies to the term "upstream" as used below. Thus, the "upstream" position in the liquid guiding system would be the reservoir, while the "downstream" position would be a position where liquid cannot be guided further, such as an end or a position where liquid is discharged from the liquid guiding system, such as an outlet. Therefore, the term "downstream" should be understood in the direction of the liquid flow, and the term "upstream" should be understood in the opposite direction of the liquid flow.

[0010] Furthermore, each interface unit constitutes a minimal liquid dispensing unit and can be multiplied within the system, comprising an injector connected to a conduit assembly, wherein each injector exhibits a body and an injection channel. Here, each injection channel includes an inlet, an outlet, and an intermediate section. The inlet receives liquid from the supply channel, the outlet dispenses liquid into the sample holder liquid inlet, and the intermediate section or guide guides the liquid from the inlet to the outlet, wherein the outlet terminates at the injector's outlet opening. Additionally, the cross-section of the inlet of each injection channel is ø进 The cross-section of the middle part ø 中间 and the cross section of the outlet. 出 The following conditions must be met: ø 出 > ø 进 And ø 出 > ø 中间 That is, the cross-section ø of the outlet of each injection channel. 出 The cross-section ø of the inlet of the corresponding injection channel is larger than the cross-section of the inlet. 进 And the cross-section ø of the outlet of each injection channel 出 The cross-section ø of the middle part of the corresponding injection channel is larger than the cross-section of the middle part of the injection channel. 中间 In this context, the term "cross-section" specifically refers to the corresponding diameter, also known as the inner diameter, of the various portions / sections / segments of the injection channel, i.e., the borehole diameter of the drilled portion / section / segment that constitutes the injection channel. This specially developed geometry of the injector channel of the interface unit of the dispensing system prevents uncontrolled leakage of liquid from the injector. As an example of such dimensional specification, the cross-section ø of the inlet portion of the corresponding injection channel... 进 The diameter can be between 0.7 mm and 0.9 mm, for example, 0.8 mm; the cross-sectional area of ​​the middle part of the corresponding injection channel is ø 中间 The diameter can be between 0.4 mm and 0.6 mm, for example, 0.5 mm; and the cross-sectional area of ​​the outlet of each injection channel is ø 出 It can be between 1.1 mm and 1.3 mm, for example, 1.2 mm.

[0011] In a typical dispensing operation, the liquid to be dispensed is conveyed from the supply channel to each interface unit, and more specifically, through the inlet to the guide section, and then further to the outlet of the injector. During the flow of liquid from the middle of the injection channel of the interface unit according to the invention to the outlet section with a larger cross-section, the pressure of the liquid flow through the injection channel decreases, and the liquid expands, resulting in improved controllability of droplet breakage at the outlet opening and preventing uncontrolled leakage of liquid from the injector. Therefore, the geometry of each injector is designed to ensure controlled droplet breakage, particularly when appropriate dispensing parameters are used for the liquid to be dispensed. This enables controlled liquid dispensing. In this regard, certain actuators of the dispensing system can be controlled by parameters, such as a liquid pump, a corresponding pressure generator, a pressure control device, a first valve, a second valve, etc. Therefore, when using the dispensing system as described above, it is possible to affect the dispensing of liquids with different viscosities and surface tensions.

[0012] As described above, by specifically annotating the dimensions of the internal structure of each injector (i.e., its injection channel), uncontrolled droplet separation, which would lead to inaccuracies in precise volume delivery, can be avoided. Therefore, the specially designed flow geometry of the liquid discharge from the injector, particularly in conjunction with the predetermined parameterization of the liquid pump and the valve arranged downstream therefrom, enables the precise, controlled dispensing of a predetermined small volume of liquid (such as silicone oil with a volume ≤ 200 µl) into the filling area of ​​the microfluidic sample holder in a desired manner. Furthermore, utilizing the specific structure of the aforementioned dispensing system of the present invention, flushing of the entire flow line from the reservoir to the outlet opening can be achieved to remove air bubbles, i.e., flushing the flow manifold and injector in a bubble-free state to avoid the formation of air bubbles and incorrect volumes during dispensing.

[0013] Generally, with the dispensing system according to the invention, the flushing / infusion process can be divided into a stage of flushing the periphery and the dispenser, and a subsequent stage of flushing the injector. In this regard, a first valve, i.e., the infusion valve, is first opened, and liquid is pumped into its return flow through the open infusion valve via the pump inlet. The first valve is then closed, thereby creating a closed column of liquid held within the supply channel. In doing so, a small air cushion may form in front of the second valve, which can be expelled during further flushing. Especially during the initial flushing, turbulence within the liquid of the initial flushing flow can trap atmospheric fluids such as air in the liquid, which can lead to the formation of an undesirable air / liquid mixture within the conduit components of the dispensing system. Such air / liquid mixtures are particularly problematic because any bubbles generated therefrom and dispensed by the injector may burst at the outlet, thus contaminating the periphery of the injector. During liquid dispensing, bubble bursting will be noticeable due to the generation of inaccurate droplet breakage behavior and thus the dispensing of incorrect volumes. However, with the structure of the dispensing system of the invention, potentially problematic air / liquid mixtures can now be safely removed through the return flow flowing through the open infusion valve. Only after this can the injector be vented / filled, free from contamination by potentially bursting air bubbles. Therefore, after the supply channels have been flushed and all potential air bubbles have been removed, each injection channel can be individually and continuously connected to the liquid column within the supply channel by opening the corresponding second valve (i.e., the on / off valve of the corresponding interface unit). In doing so, any unwanted cavitation such as air bubbles remaining in the system can be expelled one by one through the injectors. This allows for controlled cascade filling. For the initial flushing of the system, a collection container can be placed below the dispensing system (i.e., below each injector) to collect the flushed liquid. Such a collection container can be implemented as a collection tray, from which the flushed and collected liquid can be pumped or directed to a waste container. Alternatively, an empty microfluidic sample holder can be placed below the dispensing system to collect the flushed liquid, and then the microfluidic sample holder can be discarded.

[0014] Regarding the liquid to be dispensed, it has been shown that the liquid can be a sealing fluid used to seal the sample within the micropores of the corresponding microfluidic sample holder. For example, the silicone oil can be fluorosilicone oil or fluorinated silicone oil, exhibiting desired properties such as those required for optical quality, because the used sealing fluid must provide a certain degree of light transmittance for optical analysis of the sample within the micropores of the microfluidic sample holder using a camera system in subsequent stages. It must be noted here that fluorosilicone oil has a high viscosity. Therefore, if fluorosilicone oil is aspirated with a small tube diameter and excessively high suction speed, there is a risk of degassing in the oil, forming microbubbles that may interfere with subsequent processes, especially in optical applications. To prevent such degassing, the fluorosilicone oil should be aspirated by a liquid pump at a low suction speed. However, with the present invention, any air bubbles can be flushed out of the system, thereby reducing any undesirable properties of the fluorosilicone oil.

[0015] Furthermore, as another embodiment of the distribution system of the present invention, the cross-section of the inlet portion of each injection channel is ø 进 The cross-section of the middle part ø 中间 and the cross section of the outlet. 出 The following conditions must be met: ø 出 > ø 进 > ø 中间 Therefore, the liquid is transported from the inlet section with a larger cross-section to the middle section with a smaller cross-section, thus increasing the back pressure due to the reduced cross-section. Here, as an example of such dimensional calibration, the cross-section ø of the inlet section of the corresponding injection channel is... 进 The diameter can be between 0.7 mm and 0.9 mm, for example, 0.8 mm; the cross-section of the middle part of the corresponding injection channel is ø 中间 The diameter can be between 0.4 mm and 0.6 mm, for example, 0.5 mm; and the cross-sectional area of ​​the outlet of each injection channel is ø 出 It can be between 1.1 mm and 1.3 mm, for example, 1.2 mm. Additionally or alternatively, the length l of the inlet portion of each injection channel... 进 The length of the middle part l 中间 and the length of the outlet section l 出 Condition l can be satisfied 中间 ≥ l 出 One of them can also satisfy condition l 进 ≥ l 中间 >l 出 As an example of this longitudinal dimensioning, the length l of the inlet... 进 It can be between 3 mm and 5 mm, for example, about 4 mm; the length of the middle part l 中间It can also be between 3 mm and 5 mm, for example, about 4 mm; and the length of the outlet section l 出 The length can be between 1.5 mm and 2.5 mm, for example, about 2 mm. In this document, the term "length" referring to a portion of the injection channel should be understood as the length of the extension of the corresponding portion of the injection channel along its longitudinal axis (i.e., along the longitudinal axis of the injector body). With the optional structural features of each interface unit mentioned above, the volume delivery by means of the dispensing system described above can be further improved, thereby further reducing or completely avoiding uncontrolled droplet separation, and thus avoiding any inaccuracies in precise volume delivery.

[0016] Based on the dispensing system of the present invention, the end face of the injector body at the outlet opening can be maintained at a production-related minimum to reduce the contact surface of the liquid to be dispensed from the outlet opening. This further prevents the sealant from spreading to the outside of the injector. Alternatively or additionally, the inner circumference of the downstream end of the outlet portion of each injection channel may include an enlarged cross-section ø 出 The outlet chamfer, also known as the outlet bevel or phase at its downstream end, can be 40° to 50°, such as 45°. This type of outlet chamfer further prevents the sealant from spreading to the outside of the injector. Furthermore, with the dispensing system of the present invention, a channel chamfer can be provided between the inner circumference of the inlet portion and the inner circumference of the middle portion of each injection channel, this channel chamfer can be approximately 28°. This specific geometry allows the fluid flow from the larger cross-section inlet portion to the smaller cross-section middle portion to be smoothed, thereby increasing back pressure due to the reduced cross-section, while simultaneously avoiding sharp pressure peaks.

[0017] As an alternative embodiment of the dispensing system of the present invention, the body of each injector may be made of a plastic material exhibiting low surface energy, such as polytetrafluoroethylene (PTFE). In this respect, apart from the structural problems of previously proposed injectors (the inappropriate dimensional specification regarding the generally excessively large cross-section of the internal channels), the materials proposed for injectors in the past have always exhibited unfavorable surface properties. As confirmed by the inventors of the present invention, with the previously proposed injectors, each time a droplet volume is formed by such injectors, a portion of that volume spreads upwards and contaminates the injector due to surface energy and surface roughness. In this respect, it should be considered that as surface tension decreases, the contact angle between the liquid and the surface also decreases, which similarly increases wetting capacity. Therefore, to minimize this so-called extended wetting, the injector is made of a material with very low surface energy, such as PTFE or a fluoropolymer material; that is, the injector of the present invention may be made of a special plastic that itself prevents liquid from remaining on the surface of the injector, thereby further improving the ability of the dispensing system of the present invention to precisely dispense preset small volumes of liquid, such as non-polar silicone oil, in a controlled manner, ≤ 200 µl.

[0018] In another alternative embodiment of the dispensing system of the present invention, the dispensing system may further include an air supply unit for providing pressurized or compressed air to each flow channel of the microfluidic sample holder. The air supply unit may include a pressurized air reservoir, a pressure regulator, and an air supply channel connected to an air outlet nozzle. Additionally, the air outlet nozzle may be arranged adjacent to the outlet opening of the injector. Thus, air and liquid can be supplied to the sample holder by the dispensing system through at least one universal interface unit. Therefore, each interface unit provides switchable liquid delivery and controlled compressed air delivery. Overpressure can thus be generated above the filling area of ​​the sample holder to guide the liquid volume through one or more flow channels of the sample holder. By applying uniform pressure, the sealing fluid can be continuously forced through the flow channels of the microfluidic sample holder to achieve a uniform distribution of the liquid throughout the reaction path of the microfluidic sample holder. In this regard, to generate a constant, pulsation-free target pressure, a supply pressure is first applied to an accumulator, such as the reservoir mentioned above, and then connected to a pressure regulator that adjusts the target pressure to be delivered to the sample holder. In other words, the injection and pressurization are locally combined and arranged above the filling area of ​​the sample holder. Therefore, with this dispensing system, it is possible to switch from liquid delivery to pressurization without time delay. Furthermore, with this dispensing system, which may include multiple interface units, multiple channels of the sample holder can be supplied simultaneously.

[0019] To prevent any pressure loss or cross-contamination, the connection between the liquid outlet opening / air outlet nozzle and the microfluidic sample holder can be sealed. This sealed connection between the microfluidic sample holder and the dispensing system maintains the target pressure in the holder's filling area. Therefore, a sealing member can be attached to the conduit member, simultaneously surrounding the injector's outlet / outlet opening and the air outlet nozzle of each interface unit, to seal any liquid or air between the conduit member and the microfluidic sample holder to prevent leakage to the outside. In the case of multiple interface units and air outlet nozzles, the sealing member can be implemented as an elongated sealing member, such as a rod-shaped member, having a corresponding plurality of orifices, each orifice forming a combination surrounding the outlet opening and air outlet nozzle of an interface unit. Thus, generally, the sealing member can be a sealing body whose inner bore tapers substantially partially towards the microfluidic sample holder, surrounding various combinations of outlet openings and air outlet nozzles. Regarding longitudinal extension, the air outlet nozzle of each interface unit can be arranged within the inner bore of the sealing body, while the injector's outlet opening can be arranged outside the sealing body, i.e., protruding outward from the sealing body towards the microfluidic sample holder.

[0020] In another alternative embodiment of the dispensing system of the present invention, the liquid pump may include a controllable volume replacement actuator (such as a stepper motor) and a switching valve (such as a solenoid valve) for switching the delivery direction of the liquid pump. For such a bidirectional liquid pump, the liquid pump connected to the reservoir may also be referred to as a metering pump, as it can be used to meter the volume of liquid to be pumped. Here, the liquid pump can be implemented as a pump capable of drawing a specific maximum volume of liquid to be pumped. This can be achieved through volume replacement in the pump head of the liquid pump. Thus, after dispensing, the liquid pump reverses by a few microliters to draw back any droplets that may remain at the injector outlet opening. The liquid pump can then draw its maximum permissible volume and release it in multiple dispensing steps. To achieve efficient operation of the pump, a controllable volume replacement actuator can be used to set various parameters, such as dispensing speed, acceleration slope of the pump head, and / or deceleration slope. Furthermore, by using an electromagnetic switching valve as the pump valve to switch the pump flow direction, the pump flow can be reversed without opening a second valve / switching valve, resulting in the volume to be extracted on the delivery side. This significantly facilitates controlled droplet disconnection at the outlet opening of the corresponding interface unit. Moreover, the delay between pump movement and valve switching can be adapted to the corresponding process. Therefore, with this type of liquid pump, not only is the liquid to be dispensed separated in the various channels of the sample holder, but the entire system can also be flushed before the actual dispensing process begins.

[0021] In other words, to ensure constant and precise liquid volume distribution, the design of the flow channel and distribution geometry is combined with the parameterization of the actuators required for the medium flow and the monitoring of the medium flow. This enables the pumping of sealing fluids with kinematic viscosities up to 100 mm² / s and surface tensions up to 20 mN / m without bubbles, and the precise distribution of these sealing fluids. Therefore, this invention focuses primarily on the distribution system of the connectable microfluidic sample holder described above. Its filling can be controlled by different functional components: a liquid dispenser, a compressed air dispenser, and a process monitoring device. The liquid dispenser delivers a preset volume of sealing fluid to the filling area of ​​the sample holder. The compressed air dispenser applies overpressure above the filling area of ​​the sample holder after the preset volume of sealing fluid has been delivered to displace the volume of the sealing fluid and generate a constant flow that allows the medium to move through the sample holder. The process monitoring device monitors the flow rate of the constant flow so that the flow can be interrupted in time when system limits are reached. This is achieved by monitoring the yield points at multiple stages of the filling / dispensing process. One advantage of this invention is that the dispensing system of this invention offers great flexibility in relation to the physical variables of the corresponding medium to be dispensed, thereby directly influencing the type of delivery and transport in the sample holder. Furthermore, to achieve precise volumetric delivery, a complete hydraulic column is formed in the system of this invention, meaning that no cavitation or air bubbles are generated due to the removal of initial air bubbles from the system. In this respect, it may be advantageous that the reservoir (also called a storage tank) is located at a similar level or below the liquid pump, and branches may be located behind the reservoir. With this arrangement, a first line is connected to the suction side of the liquid pump, and one end of a second line is arranged above the reservoir as a discharge port. Due to the position of the reservoir relative to the liquid pump, the reservoir is prevented from being emptied by the liquid pump when the dispensing system is closed and the on / off states of the valves and the liquid pump are uncertain. Additionally, the liquid level in the reservoir can be determined by calculating a predetermined pump stroke. Alternatively or additionally, the reservoir may also be equipped with a capacitive sensor system by which the liquid level in the reservoir can be determined. For example, a capacitive sensor system may include an electrode attached to a nozzle extending through the reservoir, and another electrode implemented through the ground of the reservoir, wherein the electric field between the two electrodes can be measured. Thus, as the liquid level decreases, the change in capacity can be measured, reflecting the filling level of the reservoir. Therefore, the liquid level can be continuously monitored.

[0022] According to another aspect of the invention, a microfluidic sample holder sealing system is provided for filling a microfluidic sample holder with a pre-defined small volume of sealing fluid of ≤ 200 µl. More specifically, the microfluidic sample holder sealing system includes a dispensing system as described above and a microfluidic sample holder, such as a disposable microfluidic sample holder, which is a consumable and can be implemented as a microfluidic device such as a microfluidic chip, also known as a digital polymerase chain reaction (dPCR) chip. The microfluidic sample holder includes a filling region, an outlet region, and at least one flow channel (preferably multiple flow channels), wherein each flow channel exhibits a liquid inlet connected to the outlet region via a corresponding flow channel, wherein the liquid region (i.e., each liquid inlet) can be pre-filled with sample liquid before the introduction of the sealing fluid, for example, by means of removing sample liquid and adding it to the respective liquid inlet. Furthermore, a monitoring device can be provided for each flow channel of the microfluidic sample holder, wherein the monitoring device is used to monitor the filling of the corresponding flow channel. For example, such a monitoring device can be disposed separately from the microfluidic sample holder, for example, arranged on a sensor circuit board, etc., and can be placed adjacent to the flow channel of the microfluidic sample holder. More specifically, the microfluidic sample holder can be arranged on a separate sensor circuit board, and can even be actively pressed against the sensor circuit board during filling. Thus, a monitoring device located on the sensor circuit board can record capacitance changes, for example, when the microfluidic sample holder is inserted into the microfluidic sample holder sealing system of the present invention, or when a microfluidic sample holder already filled with a main mixture, etc., is inserted, or when the microfluidic sample holder is finally filled with a sealing liquid. Here, the monitoring device may include at least one capacitive sensor formed by two electrodes arranged to contact the bottom side of the microfluidic sample holder at the corresponding flow channel to be monitored. Thus, process monitoring can be performed by means of a capacitive sensor located below the consumable and in direct contact with its bottom side, causing the reaction path of the microfluidic sample holder in the form of a flow channel to be subdivided into segments by the capacitive sensor. For example, several electrodes may be arranged below each flow channel of the microfluidic sample holder, wherein a pair of opposing electrodes provides a capacitive sensor, and each capacitive sensor divides the flow channel into two segments. Based on this, one capacitive sensor can divide the flow channel into two sections, two capacitive sensors can divide the flow channel into three sections, three capacitive sensors can divide the flow channel into four sections, and so on.

[0023] With an electric field generated between the two electrodes as described above, any liquid flowing within the sample holder will act as a dielectric and change the capacitance of each capacitive sensor, which can be measured. By means of, for example, four separate capacitive sensors in each reaction path, the filling level and filling process can be monitored via four different thresholds in the form of four separate d-capacitive sensors arranged along the flow channel. Therefore, process monitoring as described above is used to ensure filling not only indirectly through the parameterization of all involved actuators, but also directly. With this structural feature, different filling levels can be recorded before and during the filling process of the microfluidic sample holder, leading to different conclusions:

[0024] • Sample present in microfluidic sample holder: If a microfluidic sample holder is used to provide the sample, at least one capacitive sensor should deviate from its original state in terms of capacitance. If no capacitive sensor deviates from its original state in terms of capacitance, it can be inferred that no sample is present in the sample holder. Therefore, since the sealing fluid to be dispensed is only used to seal the sample within the microfluidic sample holder, it is not necessary to seal the microfluidic sample holder. In such cases, a used microfluidic sample holder can be identified as an empty sample holder or "misfilled".

[0025] • Filling rate / volume flow rate can be determined: The presence of a flow front during filling with sample liquid and / or sealing fluid can be detected through several measurement points provided by multiple capacitive sensors arranged along each flow channel. Using determined time values, the filling rate / volume flow rate in the monitored microfluidic sample holder's flow channels can be calculated; or

[0026] • Reaching the end of the reaction distance: The last detection section, where the last capacitive sensor located downstream of the monitored flow channel, determines whether liquid has reached the end of the reaction path. In addition to determining the time parameter for the end of a specified filling process, the liquid filling can also be directly terminated using the sensor signal from the last capacitive sensor.

[0027] The above list of possible conclusions should not be construed as a complete list, but rather as a representation of several possible conclusions. Of course, based on the measurement results, other possible conclusions can be drawn within the scope of this invention.

[0028] According to another aspect of the invention, a method is provided for dispensing a sealing fluid into a pre-filled microfluidic sample holder using the dispensing system as described above. More specifically, and particularly concerning the use of Mirasil DM50 as the sealing fluid, the method of the invention comprises the following steps:

[0029] (a) Open the first valve of the aforementioned distribution system;

[0030] (b) The supply channel is flushed with sealing fluid by pumping sealing fluid into it, wherein excess sealing fluid that may contain air bubbles can be discharged through an open first valve;

[0031] (c) Close the first valve, thereby creating a closed hydraulic seal column in the supply channel;

[0032] (d) Open the second valve individually and sequentially to connect each injection channel to the supply channel and flush each injector with sealing fluid;

[0033] (e) Pump more sealing fluid into the supply channel, where excess sealing fluid, which may include air bubbles, can be discharged through the corresponding injector, where an empty microfluidic sample holder can be used to capture the excess sealing fluid, and then the microfluidic sample holder can be discarded and the second valve closed.

[0034] (f) A microfluidic sample holder is arranged at the dispensing system, the microfluidic sample holder including a filling area, an outlet and at least one flow channel, the filling area being prefilled with sample liquid and the prefilled microfluidic sample holder being filled with a sealing liquid, which is aligned with each flow channel in each injection unit.

[0035] (g) Open the second valve again, and then pump a preset volume of sealing liquid (e.g., between 90 µl and 120 µl, such as 105 µl) through the supply channel into the filling area of ​​the microfluidic sample holder, wherein the time delay between opening the second valve and dispensing the liquid into the filling area of ​​the microfluidic sample holder may be about 0 s or exactly 0 s, wherein the preferred dispensing rate is between 180 µl / s and 220 µl / s, such as 200 µl / s;

[0036] (h) Close each second valve immediately after the volume delivery is completed to achieve a constant droplet break at the outlet opening of each injector, wherein the valve should be closed immediately or almost immediately after the liquid pump delivers the volume so that the droplet break remains constant and no dripping occurs, resulting in a time delay of approximately 0 ms or exactly 0 ms between the dispensing action and the closure of each second valve;

[0037] (i) A liquid pump draws sealing fluid from the supply channel (also known as re-soaking) and opens the first valve for overpressure compensation, wherein a slight overpressure may be generated in the system due to the rapid closure of the second valve, which can be compensated by the liquid pump drawing a small amount of liquid back into the system in the opposite direction, and by opening the first valve for additional overpressure compensation for a short period thereafter; wherein the time delay between closing each second valve and re-soaking may be between 40 ms and 60 ms, such as 50 ms; the volume to be re-soaked may be between 2 µl and 5 µl, such as 3 µl; the pump speed for re-soaking may be between 20 µl / s and 40 µl / s, such as 30 µl / s; and the time delay between the end of the re-soaking process and opening the first valve may be approximately 0 ms or exactly 0 ms;

[0038] (j) Close the first valve, wherein the time delay between opening and closing the first valve may be between 900 ms and 1100 ms, such as 1000 ms;

[0039] (k) Open the second valve individually and sequentially to connect each injection channel to the supply channel; and

[0040] (l) More sealing fluid is drawn from the supply channel by a liquid pump to remove any residual sealing fluid that may remain at the injector outlet opening. This means that the second valve upstream of each injector is reopened in the previous step, so that no droplets remain at the injector outlet opening, which may have been caused by the previous droplet break, and so that the liquid can be pumped back into the system by the liquid pump in the opposite direction to the dispensing direction. The time delay between opening the second valve and the corresponding re-soaking can be about 0 ms or exactly 0 ms; the volume of liquid to be re-soaked can be between 3 µl and 8 µl, such as 5 µl; and the pump speed for re-soaking can be between 20 µl / s and 40 µl / s, such as 30 µl / s.

[0041] Then, following the steps described above, each second valve located upstream of the respective injector can be closed again, with the delay between step (l) and closure potentially between 900 ms and 1100 ms, such as 1000 ms. Furthermore, to return the liquid pump to its initial position, the switching valve on the liquid pump can be switched to its suction side (i.e., opposite to the dispensing side), and the liquid pump moved to its zero position, allowing residual liquid to return to the reservoir. The switching valve of the liquid pump can then be switched back to the delivery / dispensing side. Additionally, in the initial stage, prior to step (a) above, the liquid pump may have been filled with liquid, i.e., the liquid pump draws liquid from the reservoir, advantageously at a low speed to prevent degassing. This can be achieved by switching the switching valve of the liquid pump to the suction side and drawing in the required volume of liquid. The switching valve of the pump is then switched back to the delivery or dispensing side, i.e., towards the conduit assembly and ultimately towards the pumping direction of the injector. Therefore, an aspiration rate of approximately 40 µl / s and a time delay of approximately 2050 ms between aspiration and opening of the switching valve are advantageous, allowing the oil column in the tubing time to reduce any resultant force caused by aspiration. Using this dispensing method, the sealing fluid can be successfully dispensed into the filling area of ​​the corresponding microfluidic sample holder in a bubble-free form with no residue at the outlet opening of each injector. Based on this, it becomes particularly clear how injector geometry and appropriate parameter settings can be used to ensure that the discharged fluid is free from any volumetric deviation.

[0042] The above method can be divided into different stages, namely steps (a) to (e): opening the first valve, flushing the supply channel with sealing fluid, closing the first valve, opening the second valve individually and sequentially, pumping more sealing fluid into the supply channel, and closing the second valve again constitute the initial filling stage or filling process of the distribution system. The remaining steps (f) to (l) constitute the filling stage or filling process of filling the corresponding microfluidic sample holder with sealing fluid. During the filling stage, the filling of each flow channel can be monitored by a corresponding monitoring device, as detailed above. Additionally, for each subsequent pre-filled microfluidic sample holder, the filling phases of steps (f) to (l) above can be repeated. However, performing the initial infusion phases of steps (a) to (e) only before starting a batch of microfluidic sample holders to be sealed is sufficient, for example, when the dispensing system is opened, or at certain time intervals (e.g., once or twice a day), or after a certain number of sealed microfluidic sample holders, or after the microfluidic sample holder sealing system has been unused for a period of time, i.e., typically at certain times when the system needs to be flushed to ensure system cleanliness.

[0043] In the implementation of the method of the present invention as described above, in addition to the above-described method steps, optional additional method steps may be performed, namely, applying air overpressure above the filling area of ​​the microfluidic sample holder by means of the air supply unit of the dispensing system, thereby distributing the sealing liquid, for example, into the filling area through each flow channel of the microfluidic sample holder at a constant distribution flow rate. In doing so, the sample liquid pre-filled into the filling area of ​​the microfluidic sample holder before the sealing liquid is dispensed into the filling area of ​​the microfluidic sample holder is propelled through each flow channel of the microfluidic sample holder by means of the sealing liquid, a process driven by the constant distribution flow rate provided by the overpressured air. Therefore, the applied air pushes the sealing liquid dispensed into the filling area of ​​the microfluidic sample holder and through each flow channel of the microfluidic sample holder, and then the sealing liquid pushes the sample liquid through each flow channel of the microfluidic sample holder, thereby filling each micropore in the microfluidic sample holder with the sample, and subsequently sealing each micropore in the microfluidic sample holder filled with the sample with the sealing liquid.

[0044] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references. Similarly, the words “comprising,” “including,” and “covering” should be interpreted as inclusive rather than exclusive; that is, as “including but not limited to.” Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and.” The terms “multiple,” “many,” or “numerous” refer to two or more, i.e., 2 or > 2, in integer multiples, where the terms “single” or “single” refer to one, i.e., equal to 1. Furthermore, the term “at least one” should be understood as one or more, i.e., 1 or > 1, also in integer multiples. Therefore, the use of words in the singular or plural also includes both the plural and singular, respectively. Furthermore, the words “this,” “above,” “preceding,” and “below,” and words of similar meaning used in this patent application should refer to the entire patent application, and not to any particular part of this patent application. Additionally, as used herein, the term “about” refers to a quantity that is approximately, almost, or nearly equal to or less than about 5%, about 4%, about 3%, about 2%, or about 1%. It is used to indicate the same quantity or an equivalent quantity adjacent to it.

[0045] Furthermore, certain terms are used for convenience and are not intended to limit the invention. The terms “right,” “left,” “up,” “down,” “below,” and “above” refer to directions in the figures. Terms include explicitly mentioned terms and their derivatives, as well as terms with similar meanings. Additionally, spatial relative terms such as “below,” “below,” “under,” “above,” “on top,” “near,” “far,” etc., may be used to describe the relationship between one element or feature and another element or feature as shown in the figures. These spatial relative terms are intended to cover different positions and orientations of the device in use or operation, in addition to the positions and orientations shown in the figures. For example, if the device in the figures is flipped, an element described as “below” or “below” other elements or features will be “above” or “above” other elements or features. Thus, the exemplary term “below” can cover both above and below positions and orientations. The device may be oriented in other ways (rotated 90° or otherwise), and the spatial relative descriptive terms used herein will be interpreted accordingly. Similarly, descriptions of movement along and around various axes include various specific device positions and orientations.

[0046] To avoid repetition in the description of the accompanying drawings and various aspects, as well as the illustrative embodiments, it should be understood that many features are common to many aspects and embodiments. The description of specific embodiments of this disclosure is not exhaustive or intended to limit this disclosure to the precise forms disclosed. While specific embodiments and examples of this disclosure have been described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. Specific elements of any of the foregoing embodiments may be combined with or substituted for elements in other embodiments. Furthermore, although advantages associated with certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of this disclosure as defined by the appended claims. The omission of an aspect in the description or drawings does not imply that the aspect is missing in the embodiment in which the aspect is included. Rather, the aspect may be omitted for clarity and to avoid a lengthy description. In this context, the following description applies to the remainder of this specification: if reference numerals in the drawings are included in the directly relevant parts of the description for the purpose of clarifying the drawings and are not explained therein, reference may be made to previous or subsequent parts of the description. Furthermore, for clarity, if not all features of a component are provided in a portion of an accompanying drawing, reference may be made to other portions of the same drawing. Similar reference numerals in two or more drawings denote the same or similar elements.

[0047] The following examples are intended to illustrate various specific embodiments of the invention. Therefore, the specific modifications discussed below should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it should therefore be understood that such equivalent embodiments are included herein. Other aspects and advantages of the invention will become apparent from the following description of specific embodiments illustrated in the accompanying drawings.

[0048] Throughout this specification, references to "one or more embodiments" that are not within the scope of the appended claims are merely possible exemplary implementations and are therefore not part of the invention. Attached Figure Description

[0049] Figure 1 is a partially cross-sectional conceptual perspective view of a dispensing system according to an embodiment of the present invention, shown in exploded form, with a microfluidic sample holder arranged below;

[0050] Figure 2 is a partially cross-sectional conceptual side view of the dispensing system shown in exploded form in Figure 1, with the microfluidic sample holder arranged below, and includes an enlarged cross-sectional view of the interface unit according to the invention.

[0051] Figure 3 is an enlarged cross-sectional side view of the injector of the distribution system shown in exploded form in Figures 1 and 2;

[0052] Figure 4 is a conceptual perspective view of an exploded form of a microfluidic sample holder sealing system according to an embodiment of the present invention, which includes the dispensing system shown in Figures 1 and 2;

[0053] Figure 5 is a conceptual perspective view of the exploded form of the microfluidic sample holder sealing system shown in Figure 4, as viewed from above, which includes the dispensing system;

[0054] Figure 6 is a conceptual plan view of the microfluidic sample holders shown in Figures 1, 2, 4, and 5, viewed from below.

[0055] Figure 7 is a flowchart illustrating one embodiment of the method of the present invention.

[0056] List of reference numerals

[0057] 1 Liquid distribution system

[0058] 2 Interface Unit

[0059] 3. Catheter components

[0060] 31. Supply channel for catheter components

[0061] 32 liquid distribution channels

[0062] 33 Gas Distribution Channel

[0063] Port 34

[0064] 35 Liquid Connector

[0065] 4. First valve / filling valve / waste liquid valve

[0066] 5. Second valve / on / off valve

[0067] 6 injectors

[0068] 61 Injector Body

[0069] The first end face of the body of the 611 injector

[0070] The second end face of the body of the 612 injector

[0071] Shoulder of the 613 injector body

[0072] Injection channel of 62 injector

[0073] 621 injector injection channel inlet

[0074] The middle part of the injection channel of the 622 injector

[0075] The outlet of the injection channel of the 623 injector

[0076] Chamfer at the outlet of the injection channel of the 624 injector

[0077] Chamfering of the injection channel of the 625 injector

[0078] 63 Outlet opening / nozzle orifice

[0079] 7 air supply units

[0080] 71 Air outlet nozzle

[0081] 8 sealing components

[0082] 81 Inner hole of sealing component

[0083] 82 The inner edge of the inner hole of the sealing component

[0084] 9 Microfluidic Sample Holders

[0085] 91 Filling area of ​​microfluidic sample holder

[0086] Liquid inlet of the flow channel of the 911 microfluidic sample holder

[0087] Flow channels of 92 microfluidic sample holder

[0088] 921 monitoring device

[0089] 9211 Capacitive Sensor

[0090] The first electrode of the 9212 capacitive sensor

[0091] The second electrode of the 9213 capacitive sensor

[0092] 93 Microfluidic Sample Holder Outlet Area / Waste Area

[0093] Liquid outlet of the flow channel of the 931 microfluidic sample holder

[0094] 94 Microfluidic Sample Holder Bottom / Lower Side

[0095] Liquid flow direction in the 95 flow channel

[0096] 10 Microfluidic Sample Holder Sealing System

[0097] ø 进 Cross-section of the injection channel inlet of the injector

[0098] ø 中间 Cross-section of the middle part of the injection channel of the injector

[0099] ø 出 Cross-section of the injection channel outlet of the injector

[0100] Length of the injection channel of the injector

[0101] l 进 Length of the injection channel inlet of the injector

[0102] l 中间 Length of the middle part of the injection channel of the injector

[0103] l 出 Length of the injection channel outlet of the injector

[0104] P1 First Major Stage / Infusion Stage

[0105] P2 Second Main Stage / Filling Stage

[0106] S0 Preparation Stage / Preparation Steps

[0107] Methods and steps for the S1-S5 infusion phase

[0108] Method steps for the S6 – S12 filling stage

[0109] S13 Optional Additional Compressed Air Application Step

[0110] End method end Detailed Implementation

[0111] An embodiment of the dispensing system 1 according to the present invention is shown in FIG1 in a partially sectional perspective view, wherein the structure of the dispensing system 1 is shown in an exploded manner so as to allow for a more accurate description of the structural relationships between these components. Additionally, in FIG1, the microfluidic sample holder 9 is shown alongside the dispensing system 1, particularly below it, wherein filling of the microfluidic sample holder 9 by the dispensing system 1 only occurs when the microfluidic sample holder 9 is positioned below the dispensing system 1. Here, the combination of the dispensing system 1 and the microfluidic sample holder 9 constitutes the microfluidic sample holder sealing system 10 according to the present invention. To better illustrate the main components of the dispensing system 1, certain components of the dispensing system 1, such as the reservoir or the liquid pump connected thereto, are omitted in FIG1 to focus on the core concept of the invention. However, it should be noted that in the currently described embodiment, the reservoir is a sealing liquid reservoir, and therefore, the liquid pump is a sealing liquid pump, such that the dispensing system 1 of the currently described embodiment is used to dispense the sealing liquid, wherein the sealing liquid pump includes a controllable volume replacement actuator in the form of a stepper motor and a switching valve in the form of a solenoid valve.

[0112] As shown in Figure 1, and as can be seen from Figures 4 and 5, for example, the distribution system 1 includes a conduit member 3 in the form of a flat plate or plate, comprising a common supply channel 31 and several piping systems in the form of a fluid distribution system, such as a liquid distribution channel 32 connecting to the supply channel 31, thus making the supply channel 31 a "common" supply channel 31, or a gas distribution channel 33 extending separately from the supply channel 31 or the liquid distribution channel 32 through the conduit member 3. Liquid can be supplied by means of a liquid pump (not shown), such as from a reservoir (not shown) through the liquid pump (not shown) to a liquid connector 35 of the conduit member 3 to the supply channel 31, and further through the liquid distribution channel 32, wherein the liquid connector 35 serves as the hub of the liquid pump. To enable the introduction of liquid into the supply channel 31, a first valve or filling valve is connected to one end of the supply channel 31 opposite the end of the supply channel 31 associated with the liquid connector 35. Here, in order to properly introduce liquid into the supply channel 31, the first valve 4 can be opened, allowing the air or other substances that initially dominate the supply channel 31 to be removed by pumping liquid into the supply channel 31. This enables a closed liquid column to be formed within the supply channel 31 after the first valve 4 is closed again. Therefore, any potential air cushion within the supply channel 31 can be flushed out from the conduit member 3.

[0113] Furthermore, gas can be supplied from an air supply unit 7 (suggested only in Figure 5) through a gas distribution channel 33 of the conduit member 3, which includes features such as a pressurized air reservoir and a pressure regulator, as shown by the arrow pointing to the air supply unit 7 in Figure 5. Additionally, the distribution system 1 includes one or more injectors 6 made of a plastic material exhibiting low surface energy, such as PTFE, which are further detailed below in conjunction with Figure 3, and an elongated sealing member 8 having a partially tapered inner bore 81 disposed therein, with one inner bore 81 provided for each injector 6. Here, as also seen in Figure 2, the inner bore 81 of the sealing member 8 includes an inner edge 82 disposed inside the inner bore 81, which serves as an engagement edge for engagement with the port 34 of the conduit member 3. The inner bore 81 is mainly composed of two parts: an upstream portion with a constant inner diameter to accommodate the port 34, and a downstream portion adjacent to the upstream portion that tapers from the edge of the inner edge 82 toward the end face of the sealing member 8, which is oriented away from the conduit member 3. Here, the liquid distribution channel 32 leading from the supply channel 31 to the port 34, the gas distribution channel 33 guiding air to the air outlet nozzle 71 within the port 34, the port 34 itself including the air outlet nozzle 71, together with the injector 6, constitute the interface unit 2 of the distribution system 1, wherein the interface unit 2 is provided for each liquid inlet 911 of the filling region 91 of the microfluidic sample holder 9, i.e., for each flow channel 92 of the microfluidic sample holder 9. Therefore, any liquid to be distributed by the distribution system 1 is provided to each flow channel 92 of the microfluidic sample holder 9 by means of the corresponding interface unit 2.

[0114] Based on the above, the conduit component 3 connects to a reservoir (not shown) via a liquid pump (not shown), a liquid pump connector, and through a supply channel 31 to each interface unit 2. Here, to supply any liquid within the supply channel 31 to each interface unit 2, a second valve or switching valve is provided for each interface unit 2 within a liquid distribution channel 32, which leads from the supply channel 31 to the injector 6. Thus, the connection between the supply channel 31 and each injector 6 can be opened and closed as needed. Here, during infusion, the plurality of second valves 5 (only one second valve is shown in Figure 1 for better illustration) can be opened individually and continuously to flush the liquid distribution channel 32 one by one with liquid from the supply channel 31, thereby flushing out any air cushions, etc., through the corresponding injector 6 from the liquid distribution channel 32 and flushing them along with a certain residual volume of liquid into a movable collection tray (not shown), from which the flushing fluid and collected liquid can be pumped or directed to a waste container, etc.

[0115] To enable precise delivery of small volumes, such as approximately ≤ 200 µl, using the dispensing system 1, and to determine clear droplet breakage behavior, each injector 6 is specifically designed: as shown in Figure 3, each injector 6 includes a body 61 and an injection channel 62 disposed along the longitudinal axis of the body 61. This injection channel is in the form of a through-hole through which the injector 6 extends from a first end face 611 or inlet end face of the injector body 61 to a second end face or outlet end face. Here, the first end face 611 engages with the outer periphery of the injector 6, serving as an alignment device with the port 34 of the conduit member 3, so as to position the injector 6 within the port 34. Furthermore, as described from top to bottom in Figure 3, the injection channel 62 of the injector 6 has a through-hole for the first part of the inlet 621 to receive liquid from the supply channel 31, and for the middle part 622 to guide the liquid away from the inlet 621 to the outlet 623, which is used to dispense liquid into the sample holder liquid inlet 911. The outlet 623 of the injection channel 62 ends at the outlet opening 63 of the injector 6, from which droplets of liquid are dispensed.

[0116] Regarding the dimensions of different portions of the injection channel 62, each portion of the injection channel 62 has a different inner diameter ø. In the case of the embodiment described herein, the inner diameter ø of the injection channel 62 is designed such that the cross-sectional area ø of the outlet portion 623 of the injection channel 62 is... 出 The cross-section ø of the inlet 621 of the injection channel 62 is larger than that of the injection channel 62.进 And larger than the cross-section ø of the middle portion 622 of the injection channel 62. 中间 Additionally, in the currently described embodiment, the cross-section ø of the inlet 621 of the injection channel 62 is... 进 The cross-section ø of the middle portion 622 of the injection channel 62 is larger than that of the injection channel 62. 中间 This is an optional feature because the cross-section ø of the inlet 621 of the injection channel 62 is... 进 Alternatively, the dimensions can be set to be similar to or equal to the cross-section ø of the middle portion 622 of the injection channel 62. 中间 In this embodiment, the cross-section of the inlet 621 is ø 进 The cross-section of the middle part is approximately 0.8 mm, with a diameter of 622. 中间 It is approximately 0.5 mm, and the cross-section of the outlet 623 is ø 出 It is approximately 1.2 mm. Therefore, condition (a) ø is satisfied. 出 > ø 进 (b) ø 出 > ø 中间 And (c) ø 出 > ø 进 > ø 中间 , because ø 出 = 1.2 mm > ø 进 = 0.8 mm > ø 中间 = 0.5 mm.

[0117] Further dimensional specifications regarding the injector 6, particularly the axial length of each portion of the injection channel 62, and the length l of the middle portion 622 of the injection channel 62. 中间 The length l of the outlet section 623 is greater than 出 The length l of the middle part 622 of the injection channel 62 中间 The length l can be optionally equal to that of the outlet section 623. 出 Furthermore, in the currently described embodiment, the length l of the inlet portion 621 of the injection channel 62 is... 进 The length l of the middle part 622 is greater than or equal to 中间 The length l of the outlet portion 623 of the injection channel 62 出 The length l shorter than the middle part 622 中间 Or the length l of the inlet 621 进 Specifically, in the currently described embodiment, the outlet 623 of the injection channel 62... 出It can be approximately 2 mm or greater than 2 mm. In this embodiment, the length l of the inlet portion 621 is... 进 The length of the middle part 622 is approximately 4 mm, such as 4.12 mm. 中间 It is approximately 4 mm, such as 4.08 mm, and the length of the outlet portion 623 is l. 出 The length is approximately 2 mm, resulting in a total length of approximately 10.2 mm for the body 61 of the injector 6 and the resulting injection channel 62, where condition (d) is satisfied. 中间 ≥ l 出 (e) l 进 ≥ l 中间 > l 出 and (f) l 出 ≥ 2 mm, because l 进 = 4.12 mm ≥ l 中间 = 4.08 mm > l 出 = 2 mm.

[0118] Furthermore, regarding the outer periphery of the outlet portion 623 of the main body 61 relative to the inner diameter of the outlet portion 623 of the main body 61 (i.e. relative to the cross-section ø) 出 Based on manufacturing feasibility, the second end face 612 of the injector 6 body 61 at the outlet opening 63 is kept to a minimum size to reduce the contact surface for dispensing the sealing fluid from the outlet opening 63, thereby significantly improving droplet breakage. In this regard, as shown in Figure 3, the outer diameter of the injector 6 at the outlet 623 is smaller than the outer diameters of the injector 6 at the inlet 621 and the intermediate portion 622, where, considering manufacturing requirements, a certain thickness of injector material must be provided around the outlet 623 of the injection channel 62. For example, when drilling a hole in the body 61 of the injector 6 to introduce the injection channel 62 into the injector 6, a certain outer wall thickness is required around the injection channel 62, and this thickness must also be maintained to ensure a certain level of durability for the injector 6. Drilling a hole in the body 61 is only one of several possibilities for manufacturing the injector 6 regarding the introduction of the injection channel 62 into the injector 6.

[0119] Furthermore, when the injection channel 62 is introduced into the body 61 of the injector 6, the inner periphery of the downstream end of the outlet portion 623 of the injection channel 62 includes an outlet chamfer 624, which enlarges the cross-sectional area ø of the downstream end of the injection channel 62. 出In the currently described embodiment, the outlet chamfer 624 exhibits an angle of 45°. Here, the outlet chamfer 624 also helps to reduce the wall thickness around the downstream end of the injection channel 62, thereby achieving improved droplet breakage. Furthermore, in this embodiment, where the injection channel 62 is drilled into the body 61 of the injector 6, a channel chamfer 625 is provided between the inner circumference of the inlet portion 621 and the inner circumference of the intermediate portion 622, exhibiting an angle of approximately 28°. However, the channel chamfer 625 serves a different purpose, namely, to improve the flow of sealing fluid from the inlet portion 621 of the injection channel 62 to the intermediate portion 622. Conversely, no further chamfering is provided between the middle portion 622 and the outlet portion 623 of the injection channel 62, resulting in a clear rectangular step for sealing the liquid between the middle portion 622 and the outlet portion 623. This leads to the expansion of the liquid flow through the injection channel 62 at this point, and as the liquid expands, the flow pressure suddenly decreases, resulting in improved controllability of droplet breakage at the outlet opening 63 and preventing uncontrolled liquid discharge from the injector 6. Finally, when further observing the outer periphery of the body 61 of the injector 6, it can be clearly seen from any of Figures 1 to 5, and especially from the enlarged view in Figure 2, that a shoulder 613 protrudes from the body 61 of the injector 6, which serves as a flange for engaging with the tool used to press the injector 6 into the port 34.

[0120] from Figure 1And 2. In particular, from the enlarged view of FIG. 2, it can be seen that the exemplary interface unit 2 consists of a liquid dispensing channel 32, a gas dispensing channel 33, a port 34 (including an air outlet nozzle 71), and an injector 6. Here, it can also be seen that each interface unit 2 is surrounded by a sealing member 8. More specifically, it can be seen that the sealing member 8 includes an inner bore 81 disposed therein, wherein one inner bore 81 is provided for each injector 6. Here, from the enlarged view in FIG. 2, and as partially described above, it can be seen that the upstream portion of the inner bore 81 surrounds the outer diameter of the port 34, and the inner edge 82 of the sealing member 8 abuts the end face of the port 34 such that the air outlet nozzle 71 is at least partially kept open. In order to position the air outlet nozzle 71 on the end face of the port 34, the gas guiding channel 33 extends through the conduit member 3 and through the port 34 to its end face opposite to the conduit member 3. Furthermore, the air outlet nozzle 71 is positioned on the end face of the port 34 such that the shoulder 613 of the injector 6 does not cover the air outlet nozzle 71 when placed inside the port 34. Therefore, in the assembled state, the air outlet nozzle 71 is arranged between the sealing member 8 and the injector 6, wherein sufficient space is maintained between the inner edge 82 of the sealing member 8 and the shoulder 613 of the injector 6 to allow sufficient air to be discharged from the air outlet nozzle 71 toward the sample holder 9. This arrangement ensures an airtight seal between the port 34 and the sealing member 8, preventing air discharged from the air outlet nozzle 71 from escaping between the port 34 and the sealing member 8. This ensures sufficient pressure buildup at the air outlet nozzle 71 relative to the overpressure generated above the filling area 91 of the sample holder 9, guiding the liquid volume through the flow channel 92 of the sample holder 9.

[0121] Furthermore, as can be seen from the enlarged view in Figure 2, the shoulder 613 and the remaining downstream portion of the injector 6 (including the outlet 623 of the injection channel 62) are arranged within the tapered downstream portion of the inner bore 81 of the sealing member 8, such that the second end face 612 or the outlet end face of the injector 6 is positioned slightly outside the sealing member 8, i.e., protruding therefrom. Therefore, regarding the longitudinal extension of the injector 6 within the sealing member 8, the air outlet nozzle 71 of the interface unit 2 is arranged within the corresponding inner bore 81 of the elongated sealing member 8, while the downstream end of the injector 6 (including the second end face 612 of the body 61 of the injector 6 and the outlet opening 63) is arranged outside the sealing member 8, i.e., protruding outward from the sealing member 8 toward the microfluidic sample holder 9. Therefore, when the filling region 91 of the sample holder 9 comes into contact with the dispensing system 1, the interface unit 2 presses against the filling region 91 of the sample holder 9, causing the end face of the sealing member 8, which guides the flow path away from the conduit member 3, to press against the upper surface of the liquid inlet 911 of the sample holder 9 in the filling region 91. In doing so, an airtight connection is formed between the internal space of each inner hole 81 of the sealing member 8 and the internal space of the corresponding liquid inlet 911 of the filling region 91, so that overpressure can be applied to the filling region 91 of the sample holder 9 by means of the air supply unit 7 and the corresponding air outlet nozzle 71, so as to guide any liquid volume inside the filling region 91 of the sample holder 9 through the flow channel 92 of the sample holder 9. Additionally, in doing so, the downstream end of each injector 6 (including the second end face 612 of the body 61 of the injector 6 and the outlet opening 63) protrudes from the sealing member 8 into the corresponding liquid inlet 911 of the sample holder 9. Therefore, any sealing fluid dispensed by the dispensing system 1 via each injector 6 can be delivered without loss to the corresponding liquid inlet 911, wherein the improved droplet disconnection behavior of the interface unit 2 of the dispensing system 1 of the present invention produces the ability to accurately deliver volumes ≤ 200 µl.

[0122] The microfluidic sample holder sealing system 10 of the present invention for filling a microfluidic sample holder 9 with a preset small volume of sealing fluid ≤ 200 µl includes the aforementioned dispensing system 1 and the microfluidic sample holder 9 itself, as can be seen, for example, in Figures 1, 2, 4, and 5. Here, as described above and particularly as shown in Figures 4 and 6, the microfluidic sample holder 9 includes a filling region 91 having a plurality of liquid inlets 911, a plurality of flow channels 92, and an outlet region 93, wherein each flow channel connects a liquid inlet 911 to a corresponding liquid outlet 931 of the outlet region 93. Typically, when using the dispensing system 1 of the present embodiment, each liquid inlet 911 of the microfluidic sample holder 9 is filled with sample liquid, and the sealing fluid to be dispensed by the dispensing system 1 has been delivered to each liquid inlet 911. Then, as described above, pressurized or compressed air can be uniformly supplied to each flow channel 92 of the microfluidic sample holder 9 by means of the dispensing system 1, thereby forcing the sealing liquid continuously through the flow channels 92 of the microfluidic sample holder 9 so as to uniformly distribute the liquid throughout the reaction path of the microfluidic sample holder 9. In doing so, the applied air pushes the sealing liquid dispensed into the filling area 91 of the microfluidic sample holder 9 and flows through each flow channel 92 of the microfluidic sample holder 9 toward the outlet area 93, whereby the sealing liquid pushes the pre-filled sample liquid through each flow channel 92, thereby filling each micropore (not shown) in the microfluidic sample holder 9 with the sample, and then sealing each sample-filled micropore with the sealing liquid.

[0123] To monitor the progress of liquid movement along the flow direction 95 within each flow channel 92, the microfluidic sample holder sealing system 10 of the present invention provides a plurality of monitoring devices 921 for monitoring the filling of the flow channels 92, as shown in FIG. 6. Here, the monitoring devices 921 are separate from the microfluidic sample holder 9, for example, the monitoring devices 921 are arranged on a separate sensor circuit board (not shown), which may be placed adjacent to the flow channel 92 to be monitored. More specifically, the microfluidic sample holder 9 may be arranged on a separate sensor circuit board (not shown), and may even be actively pressed against the sensor circuit board during filling. Thus, the monitoring devices 921 located on the sensor circuit board can record capacitance changes, for example, when the microfluidic sample holder 9 is inserted into the microfluidic sample holder sealing system 10 of the present invention, or when a microfluidic sample holder 9 already filled with a main mixture, or when the microfluidic sample holder 9 is finally filled with sealing liquid. With the aid of such monitoring devices 921, the filling level and filling process can be monitored progressively. The monitoring device 921 may consist of a plurality of capacitive sensors 9211 for each flow channel 92. In a more detailed description of this embodiment, each capacitive sensor 9211 is formed by two electrodes 9212, 9213 that generate an electric field between them, wherein the liquid passing through the two electrodes 9212, 9213 serves as the dielectric, and thus, when the capacitance of the corresponding capacitive sensor 9211 is changed, the change in capacitance can be used as a sensor signal to detect the passing liquid. To generate such a detection signal most efficiently, each electrode 9212, 9213 is arranged to be in direct contact with the bottom side 94 of the microfluidic sample holder 9 at the corresponding flow channel 92. In the embodiment shown in FIG. 6, each flow channel 92 includes four capacitive sensors 9211 arranged along its longitudinal extension, i.e., the capacitive sensors 9211 are distributed along the flow channel 92 from its liquid inlet 911 downstream to its liquid outlet 931, thereby dividing each flow channel 92 into several parts, specifically three parts. Therefore, the entry of liquid can be detected by a first capacitive sensor located at the inlet of the flow channel 92 immediately following the liquid inlet 911. The filling of the first section can be detected by a second capacitive sensor located downstream of the first capacitive sensor. The filling of the second section can be detected by a third capacitive sensor located downstream of the second capacitive sensor. Finally, the filling of the third section, and thus the entire filling of the flow channel 92, can be detected by a fourth capacitive sensor located downstream of the third capacitive sensor and at the end of the flow channel 92.Of course, if further detailed monitoring of each flow channel 92 with additional sections is required, even more capacitive sensors 9211 can be arranged in each flow channel 92. The minimum number of capacitive sensors 9211 for each flow channel 92 to be monitored is two: one capacitive sensor 9211 is arranged at the inlet of the corresponding flow channel 92 immediately following the liquid inlet 911, and the other is arranged at the end of the corresponding flow channel 92, so as to monitor the entry of liquid and the gradual completion of filling of the flow channel 92. Thus, by means of the sensor signals from the monitoring device 921, several conclusions can be drawn, such as whether the liquid (i.e., the sample or sealing liquid) is actually present in the microfluidic sample holder 9, information about the filling rate of each flow channel 92, or information about whether the entire reaction distance (i.e., the entire flow channel 92 is filled with liquid) has been reached.

[0124] Based on the aforementioned distribution system 1, an embodiment of the method for dispensing sealing fluid into pre-filled microfluidic sample holders 9 according to the present invention is shown in FIG7. As can be seen from the figure, this method includes multiple method steps, which can be divided into a preparation stage and essentially two main stages: a first main stage P1, also known as the infusion stage, wherein the first valve 4 is opened, the supply channel 31 is flushed with sealing fluid, the first valve 4 is closed again, the second valve 5 is opened individually and sequentially, and then the sealing fluid is pumped into the supply channel 31; and a second main stage P2, or filling stage, for filling the corresponding microfluidic sample holder 9 with sealing fluid, wherein the filling stage can be repeated for each microfluidic sample holder 9, and wherein during the filling stage, the filling of each flow channel 92 can be monitored by a corresponding monitoring device 921. In FIG7, the method steps of the first main stage P1 and the second main stage P2 (i.e., the method steps of the infusion stage and the filling stage) are respectively arranged within dashed boxes. Additionally, in Figure 7, possible repetitions are indicated by return arrows, where repetitions may include a separate filling stage or a filling stage combined with step S13, as further described below.

[0125] More specifically, in the initial stage, i.e., before any step of the two main stages, a preparation stage / preparation step S0 occurs, in which the liquid pump may already be filled with liquid, i.e., the liquid pump draws liquid from the reservoir, advantageously at a low speed to prevent degassing. This can be achieved by switching the liquid pump's switching valve to the suction side and drawing in the required volume of liquid. Then, the pump's switching valve is switched back to the delivery or distribution side, i.e., towards the conduit member 3 and ultimately towards the injector 6. For this purpose, a suction rate of approximately 40 µl / s is applied, and the time delay between suction and opening the switching valve is approximately 2050 ms, which allows the oil column time to reduce any resultant force caused by suction.

[0126] Following the aforementioned preparation step S0, and during the implementation of the first main stage P1 and the second main stage P2 of the method of the currently described embodiment, the method includes step S1 of opening the first valve 4 of the dispensing system 1. Furthermore, the method includes step S2, which involves flushing the supply channel 31 with sealing fluid by pumping sealing fluid into the supply channel 31 using a liquid pump, wherein excess sealing fluid, which may contain air bubbles, can be discharged through the opened first valve 4. Furthermore, the method includes step S3, which involves closing the first valve 4, thereby creating a closed column of hydraulic sealing fluid within the supply channel 31. Furthermore, the method includes step S4, which involves individually and sequentially opening the second valve 5 to connect each injection channel to the supply channel 31, and flushing each injector 6 with sealing fluid as already described above in the context of the description of the dispensing system 1. Furthermore, the method includes step S5, which involves pumping more sealing fluid into the supply channel 31, where excess sealing fluid, which may contain air bubbles, can be discharged through the corresponding injector 6 into a collection tray (not shown), from which the rinsing fluid and the collected liquid can be pumped or directed to a waste container, etc. Up to this point, method steps S1 to S5 constitute the first main stage P1 or infusion stage mentioned above, where infusion includes the dispensing system 1 of the injector 6, i.e., preparation or prefilling to prepare for the actual task of dispensing system 1.

[0127] Subsequently, during the second main stage P2 or filling stage, the method includes an additional step S6, namely, arranging a microfluidic sample holder 9 at the dispensing system 1, the microfluidic sample holder having a filling region 91 pre-filled with sample liquid, wherein each injection unit is aligned with each flow channel 92 of the sample holder 9. Furthermore, the method includes a step S7, which involves reopening the second valve 5 and pumping a preset volume (e.g., 105 µl) of sealing liquid through the supply channel 31 into the filling region 91 of the microfluidic sample holder 9, wherein there is no time delay between opening the second valve 5 and dispensing the liquid into the filling region 91 of the microfluidic sample holder 9, and wherein the dispensing rate of the sealing liquid used from each injector 6 is 200 µl / s, which is generated by pumping the sealing liquid. Furthermore, the method includes step S8, which involves immediately closing each second valve 5 after volume delivery to achieve a constant droplet break at the outlet opening 63 of each injector 6. This is achieved by immediately closing the valve after the liquid pump delivers the volume, ensuring the droplet break remains constant and prevents dripping, resulting in a 0 ms time delay between the dispensing action and the closing of each second valve 5. Additionally, the method includes step S9, which involves extracting sealing fluid from the supply channel 31 using a liquid pump, also known as re-soaking, and opening the first valve 4 for overpressure compensation. This involves rapidly closing the second valve 5, which may generate a slight overpressure within the dispensing system 1. This overpressure can be compensated by the liquid pump drawing a small amount of liquid back into the dispensing system 1 in the opposite direction, and by opening the first valve 4 for additional overpressure compensation within a short period thereafter. Here, the time delay between closing each second valve 5 and the re-soaking is 50 ms, the volume to be re-soaked is 3 µl, the pump speed for the re-soaking is 30 µl / s, and there is no time delay between the end of the re-soaking process and the opening of the first valve 4. Furthermore, in step S10, the method closes the first valve 4, wherein the time delay between opening and closing the first valve 4 is 1000 ms. Additionally, the method includes step S11, which involves individually and sequentially opening the second valve 5 to connect each injection channel 62 to the supply channel 31.Furthermore, the method includes step S12, in which a liquid pump draws more sealing fluid from the supply channel 31 to remove any residual sealing fluid that may remain at the outlet opening 63 of the injectors 6. Specifically, the second valve 5 upstream of each injector 6 is reopened in the previous step, ensuring no droplets remain at the injector's outlet opening 63, which may have been caused by a previous droplet break. This allows the liquid to be pumped back into the system by the liquid pump in the opposite direction to the dispensing direction. There is no time delay between opening the second valve 5 and the corresponding re-soaking. The volume of liquid to be re-soaked is 5 µl, and the pump speed for re-soaking is 30 µl / s. Here, step S12 constitutes the final step of the filling stage.

[0128] Then, that is, after steps S0 to S12 of the method described so far, each of the second valves 5 located upstream of the respective injector 6 can be closed again, wherein the delay between step S12 and the closure of each second valve 5 is 1000 ms. Then, in order to return the liquid pump to its initial position, the switching valve on the liquid pump can be switched to its suction side (i.e., opposite to the dispensing side), and the liquid pump can be moved to its zero position, thereby allowing the residual liquid to return to the reservoir through the liquid connector 35. Then, the switching valve of the liquid pump can be switched back to the delivery / dispensing side.

[0129] Finally, after steps S0 to S12 of the method of the present invention as described above, i.e., after the preparation stage S0, the infusion stage, and the filling stage, in addition to performing the method of the present invention, an additional step S13 may be performed. This step S13 requires applying overpressure air to the filling region 91 of the microfluidic sample holder 9 (as further described above) via the air supply unit 7 of the dispensing system 1, thereby dispensing the sealing fluid into the filling region 91 through each flow channel 92 of the microfluidic sample holder 9 at a constant distribution flow rate. In doing so, the dispensing method of the present invention as described above can be extended to a method of sealing the sample liquid within the micropores (not shown) of the microfluidic sample holder 9, because step S13 specifically uses the sealing fluid to push the sample liquid pre-filled in the filling region 91 of the microfluidic sample holder 9 through each flow channel 92 of the microfluidic sample holder 9, which is driven by the constant distribution flow rate provided by the overpressure air. Therefore, in step S13, air applied in an airtight manner from port 34 of the conduit member 3 to the liquid inlet 911 of the filling area 91 of the microfluidic sample holder 9 pushes the sealing liquid distributed in each liquid inlet 911 of the microfluidic sample holder 9 into and through each flow channel 92. The sealing liquid then, at least partially by means of capillary force, pushes the pre-filled sample liquid that has been drawn into the flow channel through each flow channel 92, thereby filling each micropore (not shown) in the microfluidic sample holder 9 with the sample, and then sealing it with the following sealing liquid.

[0130] With the aid of the above-described dispensing system 1, the above-described microfluidic sample holder sealing system 10, and the above-described corresponding dispensing method, the sealing liquid can be effectively dispensed into the filling area 91 of the corresponding microfluidic sample holder 9 in a bubble-free manner, and there is no residue at the outlet opening 63 of each injector 6. The specific injector geometry as described herein and the appropriate parameterization as described above during the implementation of the method can be used to ensure that no volume deviation occurs when dispensing the liquid.

[0131] Although the invention has been described with reference to specific embodiments, it should be understood that this description is for illustrative purposes only. Therefore, the invention is intended to be limited only by the scope of the appended claims.

Claims

1. A dispensing system (1) for dispensing a pre-defined small volume of liquid ≤ 200 µl into a filling region (91) of a microfluidic sample holder (9), the microfluidic sample holder including at least one flow channel (92), the dispensing system (1) comprising reservoir, A liquid pump, connected to the reservoir, At least one interface unit (2) is provided for each flow channel (92) of the microfluidic sample holder (9), wherein the liquid is supplied to each flow channel (92) by means of the respective interface unit (2). The conduit assembly (3) includes a supply channel (31) connecting the liquid pump and each interface unit (2). The first valve (4) is connected to the supply channel (31), and At least one second valve (5) is arranged between the supply channel (31) and the corresponding interface unit (2). in The first valve (4) is a filling valve, which is configured to flush the supply channel (31) with sealing fluid by pumping sealing fluid into the supply channel (31) when open, and excess sealing fluid containing air bubbles is discharged through the open first valve (4). Each interface unit (2) includes an injector (6) connected to the conduit member (3), wherein each injector (6) includes a body (61) and an injection channel (62) disposed in the body (61). Each injection channel (62) includes an inlet (621), an outlet (623), and a middle section (622), the inlet for receiving liquid from the supply channel (31), the outlet for dispensing liquid into the sample holder liquid inlet (911), and the middle section for guiding liquid from the inlet (621) to the outlet (623), wherein the outlet (623) terminates in the outlet opening (63) of the injector (6), and the cross-section ø of the inlet portion (621) of each injection channel (62) 进 the cross-section ø of the intermediate portion (622) 中间 and the cross-section ø of the outlet portion (623) 出 satisfy the following conditions: ø 出 > ø 进 ; and ø 出 > ø 中间 。 2. The distribution system (1) according to claim 1, wherein the cross-section ø of the inlet (621) of each injection channel (62) is... 进 The cross-section of the middle part (622) 中间 and the cross-section of the outlet (623) 出 The following conditions must be met: ø 出 > ø 进 > ø 中间 。 3. The distribution system (1) according to claim 1 or 2, wherein the length of the inlet (621) of each injection channel (62) is l 进 The length l of the middle part (622) 中间 and the length l of the outlet section (623) 出 The following conditions must be met: l 中间 ≥ l 出 。 4. The distribution system (1) according to claim 3, wherein the following condition is satisfied: l 进 ≥ l 中间 > l 出 。 5. The dispensing system (1) according to claim 3, wherein l 出 ≥ 2 mm.

6. The distribution system (1) according to claim 1 or 2, wherein The inner periphery of the downstream end of the outlet (623) of each injection channel (62) includes an enlarged cross-section ø 出 The exit chamfer (624), and / or A channel chamfer (625) is provided between the inner periphery of the inlet portion (621) and the inner periphery of the middle portion (622) of each injection channel (62).

7. The distribution system (1) according to claim 6, wherein the outlet chamfer (624) has an angle of 40° to 50°.

8. The distribution system (1) according to claim 6, wherein the outlet chamfer (624) has an angle of 45°.

9. The distribution system (1) according to claim 6, wherein the channel chamfer (625) has an angle of 28°.

10. The dispensing system (1) according to claim 1 or 2, wherein the body (61) of each injector (6) is made of a plastic material exhibiting low surface energy.

11. The dispensing system (1) according to claim 1 or 2, wherein the body (61) of each injector (6) is made of PTFE.

12. The distribution system (1) according to claim 1 or 2, wherein the reservoir is a sealing fluid reservoir for providing a high-viscosity sealing fluid.

13. The dispensing system (1) according to claim 12, wherein the sealing fluid has a thickness of up to 100 mm. 2 The kinematic viscosity is 20 mN / s and the surface tension is as high as 20 mN / m.

14. The dispensing system (1) according to claim 12, wherein the sealing fluid is silicone oil.

15. The dispensing system (1) according to claim 1 or 2, wherein the dispensing system (1) further comprises an air supply unit (7) for supplying pressurized air to each flow channel (92) of the microfluidic sample holder (9).

16. The distribution system (1) according to claim 15, wherein the air supply unit (7) includes a pressurized air reservoir, a pressure regulator and an air supply channel (33) connected to an air outlet nozzle (71) for each interface unit (2).

17. The distribution system (1) according to claim 16, wherein the air outlet nozzle (71) is arranged adjacent to the outlet opening (63) of the injector (6).

18. The dispensing system (1) according to claim 16, wherein a sealing member (8) is attached to the conduit member (3) and surrounds the outlet (623) of the injector (6) and the air outlet nozzle (71) of each interface unit (2) for sealing any liquid or air between the conduit member (3) and the microfluidic sample holder (9) to prevent it from being transferred to the outside.

19. The dispensing system (1) according to claim 18, wherein the sealing member (8) includes an inner bore (81) that tapers at least partially toward the microfluidic sample holder (9).

20. The distribution system (1) according to claim 19, wherein the air outlet nozzle (71) is disposed within the inner bore (81) of the sealing member (8), and the outlet opening (63) of the injector (6) is disposed outside the sealing member (8).

21. The distribution system (1) according to claim 1 or 2, wherein the liquid pump includes a controllable volume replacement actuator and a switching valve for switching the delivery direction of the liquid pump.

22. The distribution system (1) according to claim 21, wherein the replacement actuator is a stepper motor.

23. The distribution system (1) according to claim 21, wherein the switching valve is a solenoid valve.

24. The distribution system (1) according to claim 1 or 2, wherein The first valve (4) is connected to the downstream end of the supply channel (31). The second valve (5) is a switching valve, and / or The first valve (4) is also used as a waste liquid valve.

25. A microfluidic sample holder sealing system (10) for filling a microfluidic sample holder (9) with a preset small volume of sealing fluid ≤ 200 µl, the microfluidic sample holder sealing system (10) comprising: The distribution system (1) according to any one of claims 1-24, and A microfluidic sample holder (9) includes a filling region (91), an outlet region (93), and at least one flow channel (92). At least one liquid inlet (911) of the filling region (91) is pre-filled with sample liquid. A monitoring device (921) is provided for each flow channel (92) of the microfluidic sample holder (9), wherein the monitoring device (921) is used to monitor the filling of the corresponding flow channel (92).

26. The microfluidic sample holder sealing system (10) according to claim 25, wherein the monitoring device (921) includes at least one capacitive sensor (9211) formed by two electrodes arranged to contact the bottom side (94) of the microfluidic sample holder (9) at the respective flow channel (92).

27. A method for dispensing a sealing fluid into a microfluidic sample holder (9) using a dispensing system (1) according to any one of claims 1 to 24, the method comprising the steps of: (S1) Open the first valve (4) of the distribution system (1). (S2) The supply channel (31) is flushed with sealing fluid by pumping sealing fluid into it, wherein excess sealing fluid containing air bubbles is discharged through the opened first valve (4). (S3) Close the first valve (4), thereby generating a closed hydraulic sealing column in the supply channel (31). (S4) Open the second valve (5) individually and sequentially to connect each injection channel (62) to the supply channel (31), and flush each injector (6) with sealing fluid, and (S5) Pump more sealing fluid into the supply channel (31), through which excess sealing fluid containing air bubbles is discharged via the corresponding injector (6), and close the second valve (5) again. The method further includes the following steps: (S6) A microfluidic sample holder (9) is arranged at the dispensing system (1), the microfluidic sample holder being filled with a sealing liquid and comprising a filling region (91), an outlet region (93), and at least one flow channel (92), wherein at least one liquid inlet (911) of the filling region (91) is pre-filled with sample liquid, wherein each injection unit is aligned with the liquid inlet (911) of each flow channel (92). (S7) Open the second valve (5) and pump a preset volume of sealing liquid through the supply channel (31) into the filling area (91) of the microfluidic sample holder (9). (S8) Immediately after delivering the preset volume, each second valve (5) is closed to achieve a constant droplet break at the outlet opening (63) of each injector (6). (S9) The liquid pump draws sealing fluid from the supply channel (31) and opens the first valve (4) for overpressure compensation. (S10) Close the first valve (4). (S11) The second valve (5) is opened individually and sequentially to connect each injection channel (62) to the supply channel (31), and (S12) More sealing fluid is drawn from the supply channel (31) by the liquid pump to remove the residual sealing fluid at the outlet opening (63) of the injector (6).

28. The method according to claim 27, wherein, In another step (S13), an air overpressure is applied above the filling area (91) of the microfluidic sample holder (9) by the air supply unit (7) of the dispensing system (1), thereby distributing the sealing fluid through each flow channel (92) of the microfluidic sample holder (9) into the filling area (91) at a constant distribution flow rate, wherein the sealing fluid pushes the sample liquid pre-filled into the filling area (91) of the microfluidic sample holder (9) through each flow channel (92) of the microfluidic sample holder (9) at a constant distribution flow rate.

29. The method according to claim 27 or 28, wherein steps S1 to S5 of opening the first valve (4), flushing the supply channel (31) with sealing fluid, closing the first valve (4), opening the second valve (5) individually and sequentially, pumping more sealing fluid into the supply channel (31), and closing the second valve (5) again constitute the initial filling stage of the dispensing system (1), and wherein the remaining steps S6 to S12 constitute the filling stage of filling the sealing fluid into the corresponding microfluidic sample holder (9).

30. The method of claim 29, wherein the initial infusion phase is performed together with the discarding of any excess sealant.

31. The method of claim 29, wherein the filling phase is repeated for each subsequent pre-filled microfluidic sample holder (9).

32. The method according to claim 27 or 28, wherein the filling stage of each flow channel (92) is monitored by a corresponding monitoring device (921).

Images