Microfluidic device and microfluidic method for liquefying dry reagents, in particular for detecting pathogens

Abstract

The invention relates to a microfluidic system (100) for analysing biological samples, the system comprising a chamber (110) for pre-storage of a soluble substance (190), in particular a lyophilisate, 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 in the form of a capillary channel (125). The invention also relates to a method (500) for dissolving a soluble substance (190) in such a microfluidic system (100).

Description

[0001] Description

[0002] title

[0003] Microfluidic device and microfluidic method for liquefying dry reagents, in particular for the detection of pathogens

[0004] State of the art

[0005] Reagents for biochemical or molecular biological analyses, for example for the detection of pathogens via nucleic acid amplification (e.g. as a PCR test), are often stored in freeze-dried or lyophilized form. This has the particular advantage that active components such as enzymes can be stored without refrigeration.

[0006] When pre-storing in microfluidic systems, the problem arises of liquefying the lyophilisates reproducibly and without bubbles, since this cannot be achieved by controlled pipetting and vortexing as in a manual process.

[0007] Disclosure of the invention

[0008] Advantages of the invention

[0009] Against this background, the invention relates to a microfluidic system for analyzing biological samples, in particular a microfluidic system for detecting nucleic acid sequences, for example for detecting pathogens, in particular via nucleic acid amplification.

[0010] The microfluidic system can, in particular, be a microfluidic device, for example a microfluidic cartridge, which is processed, for example, with an analytical device, as described, for example, in documents DE 10 2016 222 075 A1 and DE 10 2016 222 072 A1. One or more substances can be stored in dried form, in particular as a lyophilisate, in the system or in the device or cartridge, in particular reagents for a biochemical reaction and / or for the reconstitution of a buffer.

[0011] The microfluidic system thus has at least one chamber for the pre-storage of a soluble substance, in particular a lyophilisate. The chamber is connected to a channel for introducing a fluid to dissolve the substance (hereinafter also referred to as a supply channel), wherein at least a portion of this channel is designed as a capillary channel. In particular, the supply channel is fluidically connected directly to the chamber via a first opening. The term "chamber" is generally understood to mean a fillable volume in the system, which can accommodate the substance to be dissolved. The chamber can, for example, have a round or square shape. An elongated chamber, which thus also represents a channel, can also be understood as a chamber within the meaning of the invention. A capillary channel is, in particular, a channel with a cross-sectional area between 100 square micrometers (pm 2 ) and 1 square millimeter (mm 2), preferably between 1000 square micrometers and 0.1 square millimeters, whereby the cross-sectional area can be, for example, circular, oval, rectangular, or trapezoidal. With an oval or circular cross-section, the maximum internal diameter, i.e., with a circular cross-section, the diameter, and with an oval cross-section, in particular the major axis or twice the major semi-axis of the ellipse delimiting the cross-section, can be, for example, between 10 and 1000, preferably between 10 and 700, very preferably between 10 and 500 micrometers.

[0012] The invention advantageously provides a microfluidic system with which a soluble substance, in particular a lyophilisate, can be reproducibly dissolved, in particular dissolved and liquefied. This advantageously allows the dissolving liquid to be applied to the soluble substance slowly and in a controlled manner, which supports well-defined and reproducible liquefaction. Furthermore, this prevents air bubbles from becoming trapped or soluble substance from being lost. By using the capillary channel, the filling process is tolerant to variations in the applied pump rate or pressure. This allows a simpler design of the pump used for filling. The pump can be connected directly to the supply channel or even directly to the capillary channel.The invention also makes it possible to reproducibly liquefy different types of soluble, particularly dried, substances or lyophilisates, such as powders, tablets or beads of different sizes.

[0013] The microfluidic system can have a vent channel connected to the chamber. This facilitates filling the chamber with fluid, especially liquid for dissolving the substance, since gas present in the chamber, such as air, can be easily displaced via the vent channel.

[0014] The ventilation channel can advantageously be arranged in the upper part of the chamber with respect to the direction of gravity when the system is used as intended. This facilitates the removal of air or air bubbles from the chamber during or after filling. According to a preferred development, the channel for introducing the fluid has a first valve. This allows the chamber with the substance to be separated from the rest of the fluidic network until the substance is to be dissolved. Preferably, at least part of the capillary channel is located between the valve and the chamber, in particular the first opening. According to a preferred embodiment, the capillary channel extends as far as a valve seat in the valve, in particular when the valve is designed as a normally closed diaphragm valve, for example. Furthermore, the ventilation channel or the opening via which the ventilation channel is connected to the chamber can have a valve.

[0015] According to a particular embodiment, the capillary channel opens directly into the chamber, particularly via the first opening. In other words, the supply channel no longer widens between the end of the capillary channel and the chamber. This supports the well-defined introduction of the fluid into the chamber, as the channel no longer widens and thus no longer influences the flow upstream of the opening. Furthermore, this reduces the dead volume remaining in the channel and the space required by the arrangement. In a preferred embodiment, the entire section of the channel for introducing the fluid between the first valve and the chamber, or even the entire channel for introducing the fluid, is designed as a capillary channel.

[0016] In an advantageous development of the invention, the capillary channel is connected at the end remote from the chamber, in particular via a T-junction, to a further channel for flushing an area in front of the remote end. The connection to the further channel can be formed either directly with the capillary channel or via a section of the feed channel that is not designed as a capillary channel. This section of the feed channel is advantageously designed to be as short as possible to prevent air bubbles from becoming trapped. The T-junction design has the advantage that, for example, the area in front of the feed channel can be pre-filled. This allows, for example, several liquids to be fed into the capillary channel one after the other without air bubbles. If the feed channel has a valve, the area of ​​the feed channel between this valve and the further channel can be designed as a capillary channel.This has the advantage of further reducing the dead volume in the supply channel. It also advantageously prevents air bubbles from accumulating in front of the valve or becoming effectively trapped between the valve and the other channel. Preferably, the entire fluidic path through the valve is designed as a capillary channel. In particular, the capillary channel thus extends from both sides to the valve seat, for example, when the valve is designed as a normally closed diaphragm valve.

[0017] The chamber and the capillary channel can be considered a single unit. According to further developments, the microfluidic system can comprise two or more such units, in particular at least one second unit. At least two such units can be fluidically connected to one another, in particular via a parallel circuit, wherein the two supply channels are connected to one another, in particular via the additional channel. A connection via a series circuit is also possible, wherein, for example, the chamber of the first unit is connected to the supply channel of the second unit, for example via the ventilation channel.In other words, the chamber and the capillary channel form a first unit, and the microfluidic system comprises at least a second unit, wherein the second unit has a further chamber and a channel connected to the further chamber, at least partially configured as a capillary channel. The first and second units are arranged fluidically parallel or serially to one another. These developments have the advantage that multiple substances can be dissolved in parallel or sequentially.

[0018] The microfluidic system or at least a region of the system comprising the chamber and the supply channel can preferably be formed as an injection-molded part.

[0019] The invention also relates to a method for dissolving a soluble substance in the chamber, wherein fluid, in particular liquid, is introduced into the chamber via the supply channel to dissolve the substance, in particular by operating a pump. Subsequently, at least a portion of the dissolved substance can be removed from the chamber, preferably through the capillary channel, in particular by applying a negative pressure to the capillary channel, for example again via one or the same pump.

[0020] Short description of the drawings

[0021] Embodiments of the invention are schematically illustrated in the drawings and explained in more detail in the following description. The same reference numerals are used for the elements shown in the various figures and have a similar effect, and a repeated description of the elements is omitted.

[0022] It shows

[0023] Figures 1 to 8 show embodiments of the system according to the invention and

[0024] Figure 9 is a flowchart of an embodiment of the method according to the invention.

[0025] Embodiments of the invention

[0026] The microfluidic systems described in the following embodiments can be designed in particular as microfluidic devices, also called lab-on-a-chips, for example as microfluidic cartridges or as parts of such devices or cartridges, wherein the cartridges can be those which can be processed with an analysis device, as described for example in the documents DE 10 2016 222 075 A1 and DE 10 2016 222 072 A1.

[0027] Figure 1 shows a basic embodiment of the invention in a first exemplary embodiment. The microfluidic system 100 comprises a microfluidic chamber 110 and a channel 120 for introducing a fluid into the chamber 110, hereinafter also referred to as a supply channel 120. A section of the supply channel 120 is designed as a capillary channel 125. A pressure or flow is applied to the supply channel 120. Liquid thus flows through the feed channel 120 and thus through the capillary channel 125 in a controlled manner into the chamber 110 and liquefies a soluble substance 190 located there. The substance 190 can in particular be a lyophilisate, for example comprising enzymes or a PCR master mix, for example for carrying out a PCR in the microfluidic system 100. The chamber 110 can, for example, have a size between 10 and 1000 microliters, for example 30 microliters.While the supply channel 120 generally has a diameter of 500 micrometers to 5 millimeters, the capillary channel 125 has, for example, a diameter between 10 and 500 micrometers, for example, 50 micrometers, and a length between 1 and 20 millimeters, for example, 5 millimeters. For a semicircular channel cross-section, the diameter refers to the width of the capillary channel; for other cross-sections, for example, rectangular or trapezoidal cross-sections, the depth and height of the capillary channel are of comparable magnitude. The system 100 can further comprise a ventilation channel 130 for the chamber 110, as shown in Figure 1a.

[0028] Figure 1b shows the embodiment of Figure 1a from an oblique perspective, illustrating that the chamber 110 can be rounded, for example oval or almond-shaped. Such rounding of the chamber 110 and also of the channels can be realized by manufacturing at least part of the system comprising the chamber 110 and the channels 120, 125, 130 as a plastic injection-molded part. In addition to the essentially planar shape shown in Figure 1b, the chamber can also be formed with a greater height, e.g., in the form of a small tube that is vertical with respect to gravity.

[0029] Figure 2 shows a second embodiment of the system 100 according to the invention, which, as a further development of the first embodiment according to Figure 1a, additionally has a microfluidic valve 121 in the supply channel 120 and / or a microfluidic valve 131 in the venting channel 130. As shown, the microfluidic valve 121 in the supply channel 120 is preferably arranged such that the capillary channel 125 is located between the valve 121 and the opening 111 (hereinafter referred to as the first opening 111) of the supply channel 120 into the chamber 110. This embodiment has the advantage that the chamber 100 with the substance 190 can be separated, and the substance 190 can be dissolved in a targeted manner in the desired process step. This functionality can also be implemented without the valve 131 in the venting channel 130.

[0030] Figure 3 shows a second embodiment of the system 100 according to the invention, which, as a further development of the first embodiment according to Figure 1a or the second embodiment according to Figure 2, additionally comprises a microfluidic pump 140 connected to the supply channel. The microfluidic pump 140, for example, implemented as a diaphragm pump, can generate a targeted flow through the capillary 125.

[0031] Figure 4 shows a third exemplary embodiment of the system 100 according to the invention, which, as a development of the previous exemplary embodiments, comprises a further channel 150, which is connected to the supply channel 120 via a T-junction 155. The further channel 150 can, as shown, have a further valve 151. Viewed fluidically in the direction of the chamber 110, the further channel 150 is located upstream of the capillary channel 125 and can thus advantageously be used to flush the area upstream of the capillary channel 125, in particular also the section 122 of the supply channel 120 which is located between the further channel 150 and the capillary channel 125 and is not constricted by capillary action. By such flushing, for example with a wash buffer, preferably located upstream in the system 100, contamination or air bubbles can be removed.

[0032] Figure 5 shows a fourth embodiment of the system 100 according to the invention, which can be viewed as a further development of the third embodiment. The chamber 110, together with the supply channel 120 and optionally the venting channel 130 and / or optionally the valves 121, 131, can be understood as a fluidic unit 160, wherein the system 100 can have several such units, which can be connected in series or in parallel. Figure 5 shows an example with two such units 161, 162, which are connected in parallel via the further channel 150. Alternatively, the two units could also be connected in series, for example via a connection of the venting channel 130 or another channel of the first unit 161 to the chamber 110 of the second unit 162. This embodiment and its variants have the advantage that several lyophilizates can be liquefied in parallel or sequentially.

[0033] Figure 6 shows a fifth embodiment of the system 100 according to the invention, which can be considered a modification of the previously described embodiments. As shown in Figure 6, the capillary channel 125 opens directly into the chamber 110. This has the advantage of reducing the dead volume of the arrangement and the space required in the microfluidic system 100. Furthermore, the capillary flow is not disrupted by a widening of the supply channel 120 before it opens into the chamber 110.

[0034] Figure 7a shows a sixth embodiment of the system 100 according to the invention, which can be considered a further development of the fifth embodiment. As shown in Figure 7a, the entire area 170, i.e., the entire section 170 of the supply channel 120, between the first valve 121 and the chamber 110, is designed as a capillary channel 125. This has the advantage of further reducing the dead volume of the arrangement and the space required. Furthermore, it prevents air bubbles from becoming trapped behind the valve 121.

[0035] The capillary channel 125 can preferably extend into the first valve 121, in particular as far as a valve seat in the first valve 121, wherein the valve 121 is preferably designed as a diaphragm valve. Such diaphragm valves usually comprise a valve body made of a rigid material with at least two fluidic channels and a flexible diaphragm, the actuation of which effects a fluidic connection or separation of the two channels. In particular, the diaphragm can rest on a valve seat separating the two fluidic channels and, by moving away from the valve seat in places, establish the fluidic connection between the two channels. The diaphragm can be actuated pneumatically via a third channel in the valve body. At least one of the two fluidic channels can be part of the capillary channel 125, which extends as far as the valve seat.Figure 7b shows an example of such a diaphragm valve 10 with a valve body 19, for example made of polycarbonate (PC), and a diaphragm 18, for example made of thermoplastic polyurethane (TPU). The valve body 19 has an inlet fluid channel 11 and an outlet fluid channel 12, which are separated from each other by a valve seat 15 and a first diaphragm side 16 of the diaphragm 18, which rests on the valve seat 15 and closes the two channels 11, 12. A third fluid channel 13 opens at the second membrane side 17 facing away from the valve seat 15. By applying a negative pressure via the third fluid channel 13, the membrane 18 can be partially sucked into the third channel 13 and thus moved away from the upper side 14 of the valve seat 15, so that fluid can be transported between the valve seat 15 and the membrane 18 from the first fluid channel 11 into the second fluid channel 12 and vice versa.Since this diaphragm valve 10 is closed in the resting state, which is characterized by the absence of externally applied forces, it is also referred to as a normally closed valve. The second fluid channel 12, as the outlet fluid channel 12, can now be formed as part of the capillary channel 125, as shown, and thus extend as far as the valve seat 15.

[0036] Figure 8 shows a seventh exemplary embodiment of the system 100 according to the invention, which can be understood as a further development of the third and sixth exemplary embodiments. As shown, the entire supply channel 120 is designed as a capillary channel 125. If 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 facing away from the chamber 110 is preferably also designed as a capillary channel 125, i.e. in particular the section 181 between the first valve 121 and the further channel 150 connected to the supply channel 120 (as shown). This has the advantage that the dead volume is further reduced. In addition, it prevents air bubbles from becoming trapped upstream of the first valve 121.The capillary channel 125 preferably extends from at least one side, preferably from both sides, to the valve seat of the first valve 125, so that the fluidic path through the first valve 125 is formed entirely as a capillary channel. If the first valve 125 is designed as a diaphragm valve, this can be, for example, as shown in Figure 7b and described above, wherein preferably both the inlet fluid channel 11 and the outlet fluid channel 12 are formed as part of the capillary channel 125 and each extend to the valve seat 15.

[0037] Figure 9 shows a flow diagram of an embodiment of the method 500 according to the invention. In a first step, fluid, in particular

[0038] Liquid is introduced into chamber 110 to dissolve substance 190. After the substance has liquefied, the liquid with the substance dissolved therein is at least partially withdrawn from chamber 110 through capillary channel 125 under vacuum. This has the advantage that a low flow rate is achieved through capillary channel 125, and the withdrawal occurs slowly and reproducibly. Furthermore, any air bubbles formed during liquefaction are retained in chamber 110.

Claims

Claims 1 . A microfluidic system (100) for analyzing biological samples, comprising a chamber (110) for pre-storing a soluble substance (190), in particular a lyophilisate, and a channel (120) connected to the chamber (110) for introducing a fluid for dissolving the substance (190), wherein at least a portion of the channel (120) is designed as a capillary channel (125) 2. Microfluidic system (100) according to claim 1, wherein the chamber (110) is connected to a ventilation channel (130).

3. Microfluidic system (100) according to one of the preceding claims, wherein the channel (120) for introducing the fluid has a first valve (121), wherein at least a part of the capillary channel (125) is located between the valve (121) and the chamber (110), in particular the first orifice.

4. Microfluidic system (100) according to one of claims 2 or 3, wherein the ventilation channel (130) has a second valve (131).

5. Microfluidic system (100) according to one of the preceding claims, wherein the capillary channel (125) opens directly into the chamber (110).

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

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

8. Microfluidic system (100) according to one of the preceding claims, wherein the capillary channel (125) on the end (123) facing away from the chamber (110) is connected to a further channel (120) for flushing an area in front of the opposite end (123).

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

10. Microfluidic system (100) according to 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) comprises at least a second unit (162), wherein the second unit (162) has a further chamber (110) and a channel (120) connected to the further chamber (110) with at least partial design as a capillary channel (125).

11. Microfluidic system (100) according to claim 10, wherein the first unit (161) and the second unit (162) are arranged fluidically parallel to one another, in particular via a common further channel (150).

12. Microfluidic system (100) according to claim 10, wherein the first unit (161) and the second unit (162) are arranged fluidically serially to each other, in particular via a connection of the ventilation channel (130) of the first unit (161 ) with the chamber (110) of the second unit (162).

13. Microfluidic system (100) according to one of the preceding claims, wherein at least a region of the system (100) comprising the chamber (110) and the channel (120) for introducing the fluid is formed 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) via the fluid introduction channel (120) to dissolve the soluble substance (190) located in the chamber (110).

15. The method (500) according to claim 14, wherein at least a portion of the dissolved Substance (190) is removed from the chamber (110) through the capillary channel (125), in particular by applying a negative pressure to the capillary channel (125).

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