Microfluidic device and microfluidic method for the liquefaction of dry reagents, in particular for the detection of pathogens

Abstract

The invention relates to a microfluidic system (100) for analyzing a biological sample, comprising a chamber (110) for pre-storing a soluble substance (190), in particular a lyophilizate, and a channel (120) connected to the chamber (110) for introducing a fluid for dissolving the substance (190), wherein at least one section of the channel (120) is configured as a capillary channel (125). Furthermore, the invention relates to a method (500) for dissolving a soluble substance (190) in such a microfluidic system (100).

Description

Background Technology

[0001] Reagents used in biochemical or molecular biological analyses (such as those used to detect pathogens via nucleic acid amplification, for example, as a PCR test) are often pre-stored in lyophilized or freeze-dried form. A particular advantage of this is that it allows for the storage of active components, such as enzymes, without the need for refrigeration.

[0002] For pre-storage in microfluidic systems, there is a problem in reproducibly and bubble-free liquefaction of lyophilized materials, as this cannot be achieved through controlled suction and vortexing as in manual processes. Summary of the Invention

[0003] Advantages of the present invention In this context, the present invention relates to a microfluidic system for analyzing biological samples, and more particularly to a microfluidic system for detecting nucleic acid sequences, for example for detecting pathogens, especially by nucleic acid amplification.

[0004] The microfluidic system can be, in particular, a microfluidic device, such as a microfluidic cartridge, which can be processed, for example, using analytical instruments, as described, for example, in documents DE 10 2016 222 075 A1 and DE 10 2016 222 072 A1. One or more substances in dry form, particularly as lyophilized products, can be pre-stored in the system, or in the device or cartridge, especially reagents for biochemical reactions and / or for reconstitution buffers.

[0005] Therefore, the microfluidic system has at least one chamber for pre-storing soluble substances, particularly lyophilized substances. The chamber is connected to a channel (also referred to below as a supply channel) for introducing fluid to dissolve the substance, wherein at least one section of this channel is constructed as a capillary channel. Specifically, the supply channel is directly fluidly connected to the chamber via a first port. "Chamber" is generally understood, in particular, as a fillable volume in the system capable of holding the substance to be dissolved. The chamber can, for example, have a round or angular shape. In the sense of the invention, an elongated chamber (which is therefore also considered a channel) can also be understood as a chamber. "Capillary channel" should be particularly understood as having a diameter of 100 square micrometers (µm). 2 ) and 1 square millimeter (mm) 2The channel has a cross-sectional area between 1000 square micrometers and 0.1 square millimeters, preferably between 1000 square micrometers and 0.1 square millimeters, wherein the cross-section can be constructed, for example, circular, elliptical, rectangular or trapezoidal. For elliptical or circular cross-sections, the maximum inner diameter (i.e., the diameter for circular cross-sections, and especially the principal axis or twice the semi-axis of the ellipse defining the cross-section for elliptical cross-sections) can be, for example, between 10 and 1000 micrometers, preferably between 10 and 700 micrometers, and particularly preferably between 10 and 500 micrometers.

[0006] The present invention provides an advantageous microfluidic system that enables the reproducible dissolution, especially melting and liquefaction, of soluble substances, particularly lyophilized materials. This is advantageously achieved by allowing the slow and controlled application of the dissolved liquid to the soluble substance, which facilitates well-defined and reproducible liquefaction. Furthermore, this avoids the trapping of air bubbles or the loss of soluble substances. By using capillary channels, the filling process is tolerant to changes in the applied pumping rate or pressure. This makes it easier to design pumps for filling. The pumps can be connected directly to the supply channel or even directly to the capillary channels. Moreover, the present invention enables the reproducible liquefaction of different types of soluble, particularly dry, or lyophilized substances (e.g., powders, tablets, or beads of different sizes).

[0007] Microfluidic systems can have ventilation channels that are connected to a chamber. This makes it easy to fill the chamber with a fluid, especially a liquid used to dissolve the substance, because the gas (e.g., air) in the chamber can be easily discharged through the ventilation channels.

[0008] When the system is used correctly, the ventilation passage can be advantageously arranged in the upper portion of the chamber with respect to the direction of gravity. This facilitates the removal of air or air bubbles from the chamber during or after filling.

[0009] According to a preferred modification, the channel for introducing fluid has a first valve. This allows the chamber containing the substance to be isolated from the remaining fluid network until the substance is dissolved. Preferably, at least a portion of the capillary channel is located between the valve and the chamber, particularly with the first port. In a preferred design, the capillary channel extends here to the valve seat in the valve, especially in the case of a valve constructed as, for example, a normally closed diaphragm valve. Furthermore, a venting channel or opening (through which the venting channel connects to the chamber) can have a valve.

[0010] According to a specific design, the capillary channel leads directly into the chamber, particularly via the first port. In other words, the supply channel is not widened between the end of the capillary channel and the chamber. This facilitates the introduction of fluid into the chamber in a well-defined manner, as the channel is no longer expanded in front of the port and therefore the flow is no longer disturbed. Furthermore, this reduces the dead volume retained in the channel and the space requirements for the arrangement of the structure. In a preferred design, the entire section of the channel for introducing fluid between the first valve and the chamber, or even the entire channel for introducing fluid, is constructed as a capillary channel.

[0011] In an advantageous modification of the invention, the capillary channel is connected, particularly via a T-shaped intersection, to another channel at its end away from the chamber, which is used to flush the area in front of the away end. This connection to the other channel can be achieved either directly to the capillary channel or via a section of the supply channel not designed as a capillary channel. This section of the supply channel is advantageously designed to be as short as possible to prevent air bubble trapping. The embodiment with the T-shaped intersection has the advantage of, for example, pre-filling the area in front of the supply channel. This allows, for example, multiple liquids to be injected sequentially into the capillary channel without air bubbles.

[0012] When the supply channel has a valve, the region between the valve and the other channel can be constructed as a capillary channel. This has the advantage of further reducing the dead volume in the supply channel. Furthermore, it advantageously prevents air bubbles from being located in front of the valve or effectively trapped between the valve and the other channel. Preferably, the entire fluid path through the valve is formed as a capillary channel. In particular, the capillary channel thus extends from both sides to the valve seat, as is the case, for example, in the case where the valve is constructed as a particularly normally closed diaphragm valve.

[0013] A chamber and a capillary channel can be understood as a unit. A microfluidic system, according to a modified design, can have two or more such units, particularly at least one second unit. At least two such units can be fluidly connected to each other, particularly in parallel, wherein the two supply channels are connected to each other, particularly via another channel. A series connection is also feasible, wherein, for example, the chamber of the first unit is connected to the supply channel of the second unit via a ventilation channel. In other words, a chamber and a capillary channel form a first unit, and the microfluidic system includes at least one second unit, wherein the second unit has another chamber and a channel connected to said other chamber, the channel being at least partially constructed as a capillary channel, wherein the first and second units are arranged in parallel or series with respect to each other in terms of fluidity. This modified design has the advantage that multiple substances can be dissolved in parallel or sequentially.

[0014] A microfluidic system or at least one region of a system, including a chamber and a supply channel, can preferably be configured as an injection molded part.

[0015] The subject matter of this invention also relates to a method for dissolving a soluble substance in a chamber, wherein a fluid, particularly a liquid, is introduced into the chamber via a supply channel for dissolving the substance, particularly by operation of a pump. Subsequently, at least a portion of the dissolved substance can be extracted from the chamber, preferably via a capillary channel, particularly by applying low pressure to the capillary channel, for example by another pump or the same pump. Attached Figure Description

[0016] Embodiments of the present invention are schematically illustrated in the accompanying drawings and described in detail in the following description. Elements shown in different figures and performing similar functions are referred to by the same reference numerals, wherein repeated descriptions of these elements are omitted.

[0017] in: Figure 1 to Figure 8 An embodiment of the system according to the invention is shown, and Figure 9 A flowchart illustrating an embodiment of the method according to the present invention is shown. Detailed Implementation

[0018] The microfluidic systems described in the following embodiments can be configured in particular as microfluidic devices, also known as lab-on-a-chip devices, such as microfluidic cassettes or components of such devices or cassettes, wherein the cassette can be processed using analytical instruments, such as those described in documents DE 10 2016 222 075 A1 and DE10 2016 222 072 A1.

[0019] Figure 1 illustrates a basic embodiment of the invention employing the first embodiment. The microfluidic system 100 includes a microfluidic chamber 110 and a channel 120 for introducing fluid into the chamber 110, also referred to hereinafter as a supply channel 120. Here, sections of the supply channel 120 are constructed as capillary channels 125. Pressure or flow is applied at the supply channel 120. Thus, liquid flows in a controlled manner through the supply channel 120 and therefore through the capillary channel 125 into the chamber 110, liquefying a soluble substance 190 therein. The substance 190 can in particular be a lyophilized product, which includes, for example, enzymes or PCR premixes, for example, for performing PCR in the microfluidic system 100. The chamber 110 can, for example, have a size between 10 and 1000 microliters, for example, 30 microliters. The supply channel 120 generally has a diameter ranging from 500 micrometers to 5 millimeters, while the capillary channel 125 has, for example, a diameter between 10 and 500 micrometers (e.g., 50 micrometers) and a length between 1 and 20 millimeters (e.g., 5 millimeters). For a semi-circular channel cross-section, "diameter" refers to the width of the capillary channel. For other cross-sections (e.g., rectangular or trapezoidal cross-sections), the depth and height of the capillary channel are on a similar order of magnitude. Figure 1a As shown, the system 100 may also have a ventilation passage 130 for the chamber 110.

[0020] Figure 1b Shown from an oblique perspective Figure 1a In one embodiment, chamber 110 is shown to be constructed with rounded corners, such as an elliptical or almond-shaped form. This rounded corner structure of chamber 110 and the channels can be achieved by manufacturing at least the system portion including chamber 110 and channels 120, 125, 130 as a plastic injection molded part. The chamber, in addition to being able to be formed into… Figure 1b In addition to the basically planar shape shown, it is also possible to take more shape in terms of height, for example, in the form of a small tube that stands vertically with respect to gravity.

[0021] Figure 2 A second embodiment of the system 100 according to the invention is shown, which is as follows: Figure 1aA modified version of the first embodiment additionally includes a microfluidic valve 121 in the supply channel 120 and / or a microfluidic valve 131 in the ventilation channel 130. As shown, the microfluidic valve 121 is preferably arranged in the supply channel 120 such that a capillary channel 125 is located between the valve 121 and a port 111 (also referred to hereinafter as the first port 111) in the inlet chamber 110 of the supply channel 120. This embodiment has the advantage that the chamber 100 containing the substance 190 can be isolated and the substance 190 can be selectively dissolved in the desired process steps. This functionality can also be achieved without the valve 131 in the ventilation channel 130.

[0022] Figure 3 A second embodiment of the system 100 according to the invention is shown, which is as an example of... Figure 1a The first embodiment or according to Figure 2 The second embodiment of the modified version additionally includes a microfluidic pump 140 connected to the supply channel. The microfluidic pump 140 (e.g., implemented as a diaphragm pump) enables targeted flow through the capillary channel 125.

[0023] Figure 4 A third embodiment of the system 100 according to the invention is shown, which, as a modification of the foregoing embodiment, includes an additional channel 150 connected to the supply channel 120 via a T-shaped intersection 155. The additional channel 150 can have an additional valve 151, as shown. Viewed fluidly toward the chamber 110, the additional channel 150 is located in front of the capillary channel 125 and can therefore be advantageously used to flush the area in front of the capillary channel 125, particularly the section 122 of the supply channel 120 located between the additional channel 150 and the capillary channel 125 that does not capillarily narrow. Such flushing (preferably using a washing buffer pre-stored in the system 100) removes contaminants or air bubbles.

[0024] Figure 5 A fourth embodiment of the system 100 according to the invention is shown, which can be considered a modification of the third embodiment. The chamber 110, together with the supply passage 120 and optionally with the ventilation passage 130 and / or optionally with valves 121, 131, can be understood as a fluid unit 160, wherein the system 100 can have a plurality of such units, which can be connected in series or in parallel with each other. Figure 5An embodiment with two such units 161, 162 is shown, which are connected in parallel via an additional channel 150. Alternatively, the two units can also be connected in series, for example by connecting the ventilation channel 130 of the first unit 161 or another channel to the chamber 110 of the second unit 162. An advantage of this embodiment or its variations is that multiple freeze-dried materials can be liquefied in parallel or sequentially.

[0025] Figure 6 A fifth embodiment of the system 100 according to the invention is shown, which can be understood as a variation of the embodiments described so far. (As in...) Figure 6 As shown, the capillary channel 125 leads directly into the chamber 110. This has the advantage of reducing the dead volume and space requirements of the arrangement in the microfluidic system 100. Furthermore, the capillary flow is not disturbed by the widening of the supply channel 120 before it enters the chamber 110.

[0026] Figure 7a A sixth embodiment of the system 100 according to the invention is shown, which can be considered a modification of the fifth embodiment. (As in...) Figure 7a As shown, the entire region 170 between the first valve 121 and the chamber 110 (i.e., the entire section 170 of the supply channel 120) is implemented as a capillary channel 125. This has the advantage of further reducing the dead volume and space requirements of the arrangement. Furthermore, it avoids trapping air bubbles downstream of the valve 121.

[0027] Here, the capillary channel 125 preferably reaches the interior of the first valve 121, particularly the valve seat within the first valve 121, wherein the valve 121 is preferably configured as a diaphragm valve. Such a diaphragm valve typically comprises a valve body made of a rigid material having at least two fluid channels and a flexible diaphragm, whose actuation causes fluid connection or separation of the two channels. Specifically, the diaphragm can be placed on the valve seat separating the two fluid channels and establish a fluid connection between the two channels by partially moving away from the valve seat. Here, the actuation of the diaphragm can be achieved pneumatically via a third channel in the valve body. Here, at least one of the two fluid channels can be part of the capillary channel 125 reaching the valve seat. Figure 7bAn embodiment of a diaphragm valve 10 is shown, having a valve body 19 (e.g., made of polycarbonate (PC)) and a diaphragm 18 (e.g., made of thermoplastic polyurethane (TPU)). The valve body 19 has an inlet fluid passage 11 and an outlet fluid passage 12, which are separated from each other by a valve seat 15 and a first diaphragm side 16 of the diaphragm 18, which abuts against the valve seat 15 and closes the two passages 11, 12 thereon. A third fluid passage 13 enters at a second diaphragm side 17 away from the valve seat 15. By applying a low pressure (Unterdruck) via the third fluid passage 13, the diaphragm 18 can be partially drawn into the third passage 13 and thus move away from the upper side 14 of the valve seat 15, so that fluid between the valve seat 15 and the diaphragm 18 can be delivered from the first fluid passage 11 to the second fluid passage 12, and vice versa. Since this diaphragm valve 10 is closed in a static state (characterized by the absence of any externally applied force), it is also referred to as a normally closed valve. As shown, the second fluid passage 12, which serves as the outlet fluid passage 12, can now be formed as part of the capillary passage 125 and thus extends to the valve seat 15.

[0028] Figure 8 A seventh embodiment of the system 100 according to the invention is shown, which can be understood as a modification of the third and sixth embodiments. As shown, the entire supply channel 120 is implemented as a capillary channel 125. In the case where the supply channel 120 has a first valve 121, the section between the first valve 121 and the end 123 of the supply channel 120 away from the chamber 110 is preferably also constructed as a capillary channel 125, particularly the section 181 between the first valve 121 and the additional channel 150 connected to the supply channel 120 (as shown). This has the advantage that the dead volume is further reduced. Furthermore, air bubbles are avoided from being trapped in front of the first valve 121. Preferably, the capillary channel 125 extends from at least one side, preferably from both sides, to the valve seat of the first valve 125, such that the fluid path through the first valve 125 is entirely formed as a capillary channel. In the case where the first valve 125 is constructed as a diaphragm valve, it can, for example, be as Figure 7b As shown and described above, the inlet fluid passage 11 and the outlet fluid passage 12 are preferably both constructed as portions of the capillary passage 125 and extend to the valve seat 15 respectively.

[0029] Figure 9A flowchart illustrating an embodiment of the method 500 according to the invention is shown. In a first step, fluid, particularly a liquid, is introduced into chamber 110 via supply channel 120 and thus via capillary channel 125 for dissolving substance 190. After liquefaction of the substance, the liquid, together with the dissolved substance therein, is at least partially extracted from chamber 110 under low pressure via capillary channel 125. This has the advantage of achieving a low flow rate via capillary channel 125 and achieving extraction slowly and reproducibly. Furthermore, bubbles that may be generated during liquefaction are suppressed within chamber 110.

Claims

1. A microfluidic system (100) for analyzing biological samples, comprising: A chamber (110) for pre-storing soluble substances (190), particularly freeze-dried substances, and a channel (120) connected to the chamber (110), the channel being used to introduce fluid for dissolving the substances (190), wherein at least one section of the channel (120) is constructed as a capillary channel (125).

2. The microfluidic system (100) according to claim 1, wherein, The chamber (110) is connected to the ventilation channel (130).

3. The microfluidic system (100) according to any one of the preceding claims, wherein, The channel (120) for introducing fluid has a first valve (121), wherein at least a portion of the capillary channel (125) is located between the valve (121) and the chamber (110), particularly with the first port.

4. The microfluidic system (100) according to any one of claims 2 or 3, wherein, The ventilation passage (130) has a second valve (131).

5. The microfluidic system (100) according to any one of the preceding claims, wherein, The capillary channel (125) leads directly into the chamber (110).

6. The microfluidic system (100) according to any one of the preceding claims, wherein, The entire section of the channel (120) for introducing fluid between the first valve (121) and the chamber (110) is constructed as a capillary channel (125), or the entire channel (120) for introducing fluid is constructed as a capillary channel (125).

7. The microfluidic system (100) according to any one of the preceding claims, wherein, The system (100) has a pump (140) for pumping fluid through the capillary channel (125) into the chamber (110), wherein the pump (140) is preferably directly connected to the channel (120) for introducing fluid or preferably directly connected to the capillary channel (125).

8. The microfluidic system (100) according to any one of the preceding claims, wherein, The capillary channel (125) is connected to another channel (120) at the end (123) opposite to the chamber (110), the other channel being used to flush the area in front of the opposite end (123).

9. The microfluidic system (100) according to claim 8, wherein, The channel (120) for introducing fluid has a first valve (121), wherein the region of the channel (120) for introducing fluid between the first valve (121) and the other channel (120) is constructed as a capillary channel (125).

10. The microfluidic system (100) according to any one of the preceding claims, wherein, The chamber (110) and the capillary channel (125) form a first unit (160, 161), and wherein the system (100) includes at least one second unit (162), wherein the second unit (162) has an additional chamber (110) and a channel (120) connected to the additional chamber (110), the channel being at least partially constructed as a capillary channel (125).

11. The microfluidic system (100) according to claim 10, wherein, The first unit (161) and the second unit (162) are arranged in parallel with respect to each other in terms of fluidity, especially through a common additional channel (150).

12. The microfluidic system (100) according to claim 10, wherein, The first unit (161) and the second unit (162) are arranged in series with respect to each other in terms of fluidity, especially through the connection of the ventilation passage (130) of the first unit (161) and the chamber (110) of the second unit (162).

13. The microfluidic system (100) according to any one of the preceding claims, wherein, The system (100) includes at least one region comprising a chamber (110) and a channel (120) for introducing fluid, configured as an injection molded part.

14. A method (500) for dissolving a soluble substance (190) in a microfluidic system (100) according to any one of the preceding claims, wherein fluid is introduced into the chamber (110) through a channel (120) for introducing fluid in order to dissolve the soluble substance (190) in the chamber (110).

15. The method (500) according to claim 14, wherein, At least a portion of the dissolved substance (190) is discharged from the chamber (110) through the capillary channel (125), particularly by applying low pressure to the capillary channel (125).

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