MICROFLUIDIC VALVE

Abstract

A microfluidic valve includes a carrier layer and a flexible membrane layer arranged on a surface of the carrier layer. The surface of the carrier layer has a valve chamber in the form of a spherical cap and a membrane formed by the flexible membrane layer covers at least the valve chamber. A plurality of microfluidic channels opening into the valve chamber are formed in the surface of the carrier layer. Moreover, an inflow channel and an outflow channel are connected to one another by a microfluidic connection channel. The connection channel and the valve chamber are positioned relative to each other in such a way that in the closed state of the membrane, a fluid can flow from the inflow channel via the connection channel into the outflow channel to bridge the valve chamber, while the at least one supply channel is closed by the membrane.

Claims

1. A microfluidic valve, comprising a carrier layer and a flexible membrane layer arranged on a surface of the carrier layer, wherein the surface of the carrier layer has a valve chamber in the form of a spherical cap and a membrane formed by the flexible membrane layer covers at least the valve chamber, wherein a plurality of microfluidic channels opening into the valve chamber are formed in the surface of the carrier layer, wherein the microfluidic channels comprise an inflow channel, an outflow channel and at least one supply channel, wherein the microfluidic channels and the membrane are formed in such a manner that the membrane can be brought into a closed state by application of pressure, in which closed state the membrane is pressed into the valve chamber in order to prevent the transfer of a fluid to be introduced from the at least one supply channel into the valve chamber, wherein the inlet channel and the outlet channel are connected to each other by a microfluidic connection channel, wherein the connection channel and the valve chamber are positioned relative to each other in such a way that in a closed state of the membrane, a fluid to be supplied can flow from the inflow channel via the connection channel into the outflow channel, while the at least one supply channel is closed by the membrane, wherein, in an open state of the membrane, a fluid to be supplied can flow from the inflow channel and/or at least one fluid to be introduced can flow from the at least one supply channel into the valve chamber, wherein a fluid located in the valve chamber is able to flow out of the valve chamber via the outflow channel, wherein a flow cross-section of the inflow channel and of the outflow channel are of the same size and the connection channel is dimensioned in such a way that its flow cross-section is substantially constant in the closed state of the membrane and corresponds to the flow cross-section of the inflow channel and outflow channel.

2. The microfluidic valve according to claim 1, wherein the connection channel extends below the valve chamber with respect to the flexible membrane layer and is open in the direction of the valve chamber.

3. The microfluidic valve according to claim 1, wherein the connection channel is formed as a channel-shaped depression in the valve chamber.

4. The microfluidic valve according to claim 1, wherein the connection channel, preferably in the region of the valve chamber, is designed to extend in an arc shape between the inflow channel and the outflow channel with respect to the flexible membrane layer.

5. (canceled)

6. The microfluidic chip, comprising a chip carrier layer and a flexible chip membrane layer applied to a surface of the chip carrier layer, wherein the chip carrier layer has a plurality of fluidic connectors and a microfluidic channel system connected to the fluidic connections is formed in the surface of the chip carrier layer, wherein microfluidic valves are provided for flow regulation of the microfluidic channel system, wherein at least one of the microfluidic valve elements is formed as a microfluidic valve according to claim 1 having one supply channel, wherein the flexible membrane layer of the at least one microfluidic valve is formed by the flexible chip membrane layer, wherein the carrier layer of the at least one microfluidic valve is formed by the chip carrier layer and the microfluidic channels of the at least one microfluidic valve are part of the microfluidic channel system, wherein the supply channel of the at least one microfluidic valve is connected to one of the fluidic connectors.

7. The microfluidic chip according to claim 6, wherein the microfluidic chip has a synthesis chamber for synthesizing an oligonucleotide and a main conduit channel connected to the synthesis chamber, wherein a plurality of the microfluidic valve elements is formed as microfluidic valves, wherein the main conduit channel is formed at least in sections by the inflow channels, outflow channels and connection channels of the microfluidic valves.

8. The microfluidic chip according to claim 7, wherein a plurality of the fluidic connectors is designed as reagent connectors for supplying reagents from reagent containers connected to the respective fluidic connectors to the synthesis chamber, wherein all reagent connectors are connected to the main channel via a respective microfluidic valve.

9. A method for using the microfluidic chip according to claim 6, in an automated synthesizing device for the synthesis of oligonucleotides of a predefinable length and sequence via of a phosphoramidite synthesis, wherein the valve position of the microfluidic valve elements of the microfluidic chip is controlled by the automated synthesizing device.

10. The microfluidic valve according to claim 4, wherein a section of the connection channel being located in the region of the valve chamber extends in an arc shape between the inflow channel and the outflow channel with respect to the flexible membrane layer.

11. The method according to claim 9, wherein the automated synthesizing device synthesizes DNA strands of a predefinable length and sequence via a phosphoramidite synthesis carried out on the microfluidic chip.

12. The method according to claim 9, wherein the valve position of the at least one microfluidic valve is controlled by the automated synthesizing device.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0037] The invention will now be explained in more detail by means of an exemplary embodiment. The drawings are exemplary and are intended to illustrate the idea of the invention, but in no way to restrict it or even to reproduce it conclusively.

[0038] The drawings show as follows:

[0039] FIG. 1 shows an isometric view of a microfluidic valve with a supply channel;

[0040] FIG. 2a shows a schematic sectional view of the microfluidic valve in an open state;

[0041] FIG. 2b shows a schematic sectional view of the microfluidic valve in an open state;

[0042] FIG. 3 shows a chip layout of a microfluidic chip in which a plurality of microfluidic valves are integrated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] FIG. 1 shows an enlarged schematic representation of an exemplary embodiment of a microfluidic valve 10 according to the invention in an isometric view. The microfluidic valve 10 consists of a carrier layer 7 and a flexible membrane layer 8 applied directly to the carrier layer 7, which membrane layer 8 completely covers an upper side of the carrier layer 7, in the present exemplary embodiment.

[0044] A plurality of microfluidic channels 1,2,3,6 and a valve chamber 5 in the form of a spherical cap are formed in a surface of the carrier layer 7 forming the upper side. These structures can, for example, be embossed into the carrier layer 7 or produced by means of a lithographic process.

[0045] In the area of the valve chamber 5, the flexible membrane layer 8 forms a flexible membrane 4, which can be pressed into the valve chamber 5 by applying pressure.

[0046] The microfluidic channels 1,2,3,6 comprise an inflow channel 1 and an outflow channel 3, as well as a supply channel 2, all of which open into the valve chamber 5.

[0047] If the microfluidic valve 10 is viewed from above, i.e. along a vertical axis, so that the outline of the valve chamber 5 appears circular, then inflow channel 1 and outflow channel 3 are arranged in the present exemplary embodiment extending along a straight line, wherein the straight line extends as a secant through the circular outline of the valve chamber 5. In this view, the supply channel 2 extends radially in the direction of the imaginary center of the circular outline of the valve chamber 5 and transversely to the straight line defined by the inflow channel 1 and outflow channel 3. In the present exemplary embodiment, the straight line defined by inflow channel 1 and outflow channel 3 encloses a right angle with supply channel 2, wherein supply channel 2 is not arranged in the circular segment separated by the secant.

[0048] In addition, a microfluidic connection channel 6 is provided, which connects the inflow channel 1 and the outflow channel 3 with each other, wherein the connection channel 6 is formed as a channel-shaped depression in the valve chamber 5, which extends through the valve chamber 5 as a secant when viewed from above. In the present exemplary embodiment, the connection channel lies on the same straight line as inflow channel 1 and outflow channel 3.

[0049] The operation of the microfluidic valve 10 is clear from FIGS. 2a and 2b. In FIG. 2a, the membrane 4 is relaxed and the valve chamber 5 is thus released. Thus, basically three fluid paths are possible: firstly, fluid from the inflow channel 1 can enter the outflow channel 3 via the released valve chamber 5, secondly, fluid from the supply channel 2 can enter the outflow channel 3 via the valve chamber 5, and thirdly, fluids from inflow channel 1 and supply channel 2 can also enter the valve chamber 5 simultaneously and flow off together via the outflow channel 3.

[0050] The supply channel 2 extends into the valve chamber 5 in the radial direction, wherein a channel depth (measured in the vertical direction from the relaxed membrane layer 8) of the supply channel 2 is greater than a depth of the valve chamber 5 at each point where the supply channel 2 ends in the valve chamber 5 in the radial direction. Thus, a sealing edge 9 is formed and it is achieved that fluid present in the valve chamber 5, which originates from the supply channel 2, can be forced back into the supply channel 2 when the membrane 4 is closed or can easily flow into the valve chamber 5 when it is opened.

[0051] As can also be seen, the connection channel 6 extends below the valve chamber 5 with respect to the membrane layer 8 and is open in the direction of the valve chamber 5.

[0052] This design of valve chamber 5, supply channel 2 and connection channel 6 ensures that the membrane 4—as shown in FIG. 2b—can be brought into a closed state by applying pressure, in which closed state the membrane 4 is pressed into the valve chamber 5 in order to prevent the transfer of a fluid to be introduced from the supply channel 2 into the valve chamber 5, while a fluid to be supplied can flow from the inflow channel 1 via the connection channel 6 into the outflow channel 3, although the valve chamber 5 is closed or filled by the membrane 4. The membrane 4 also forms an upper boundary of the connection channel 6 that is open in the direction of the valve chamber 5.

[0053] In other words, with the microfluidic valve 10 discussed, it is possible that when the membrane 4 is closed, the valve chamber 5 is bridged by the connection channel 6 to allow fluid transport from the inflow channel 1 to the outflow channel 3. Accordingly, when the membrane 4 is closed, the fluid path between the inflow channel 1 and the outflow channel 3 is open via the connection channel 6.

[0054] As can also be seen, the membrane 4 is pressed against the boundary surface of the valve chamber 5 in the closed state and thus, on the one hand, closes the supply channel 2, preferably via the circumferential sealing edge 9, and, on the other hand, prevents the direct transfer of fluid from inflow channel 1 or outflow channel 3 into the (closed) valve chamber 5.

[0055] In a section (not shown) through inflow channel 1, connection channel 6 and outflow channel 3 parallel to the vertical direction, connection channel 6 extends in an arc between inflow channel 1 and outflow channel 3.

[0056] The dimensions or the geometric design of the connection channel 6 is preferably selected so that a flow cross-section of the inflow channel 1, connection channel 6 and outflow channel 3 is essentially constant in the closed state of the membrane 4, i.e. when the membrane 4 closes the connection channel 6 upwards.

[0057] Although only one supply channel 2 is provided in the previously described exemplary embodiment, it is also conceivable to provide two or more supply channels 2 opening into the valve chamber 5 to create additional fluid paths.

[0058] FIG. 3 shows a possible layout of a microfluidic chip 14 that allows synthesis of oligonucleotides in an automated device, wherein several of the microfluidic valves of the microfluidic chip 14 are designed as microfluidic valves 10.

[0059] The microfluidic chip 14 has a plurality of fluidic connections 11, wherein those fluidic connections 11 are connected to a main conduit channel 12 of the microfluidic chip 14 via a microfluidic valve 10 for supplying a reagent necessary for synthesis. Additionally, conventional microfluidic valves may also be provided, with the main conduit channel 12 being connected to a synthesis chamber 13. The synthesis chamber 14 contains a carrier medium with linker molecules, which act as a starting point for the synthesis of the oligonucleotides. The microfluidic valves 10, and preferably the other microfluidic valves, are fully integrated into the microfluidic chip 14 and are formed integrally. This is achieved by forming the valve chambers 5 and the microfluidic channels 1,2,3,6 of the microfluidic valves 10 in a chip carrier layer and thus the carrier layer 7 is formed by the chip carrier layer. Also, the flexible membrane layer 8 of the microfluidic valves 10 is formed by a chip membrane layer.

[0060] How the flow through the fluidic connection 13 of the microfluidic chip 10 is accomplished is described in the following on the basis of the first base B1. This principle can be applied analogously to all other reagents.

[0061] The fluidic connection 11 of the first base B1 is connected to a supply channel 2 of the corresponding microfluidic valve 10. The inflow channel 1, the connection channel 6 and the outflow channel 3 are formed as part of the main conduit channel 12 of the chip 14. When the microfluidic valve 10 associated with the first base B1 is closed, fluid from fluidic connections 11 further away from the synthesis chamber 13, for example solvent SOL or reagent for activating a detritylated 5′-OH group ACT, can flow through the microfluidic valve 10 associated with the first base B1 or through the section of the main conduit channel 12 formed by this microfluidic valve 10 when the microfluidic valve 10 is correspondingly open, without this fluid mixing with the first base B1.

[0062] If the microfluidic valve 10 associated with the first base B1 is open, the first base B1 can enter the main conduit channel 12 via the supply channel 2 and the valve chamber 5 of the microfluidic valve 10 and subsequently flows through the downstream, closed microfluidic valves 10.

[0063] In order to enable the flow and to establish corresponding pressure conditions, a microfluidic valve is additionally opened, which controls a fluidic outlet connection formed as a second outlet W2. The second outlet W2 is arranged downstream of the synthesis chamber 13 in the direction of flow, so that the first base B1 flows to the second outlet W2 via the main conduit channel 12 and the synthesis chamber 13. The second outlet W2 can be connected to a waste container, for example.

[0064] The microfluidic valves and the microfluidic valves 10 are basically kept in a closed position, so that the supply channels 2 associated with the reagents are blocked and only the main conduit channel 12 is open. The closed position is achieved, for example, by a control device of an automated and programmable synthesis device applying pressure to the membranes 4 of the microfluidic valves 10 or to the microfluidic valves via a pneumatic system, such as via control lines 28. If the pressure exerted on one of the microfluidic valves or microfluidic valves 10 is reduced by the control device or if the pressurization is suspended, the corresponding microfluidic valve or microfluidic valve 10 opens and the fluidic connection between the corresponding supply channel 2 and the main conduit channel 12 is established or open.

[0065] Before the synthesis can start, it is necessary to supply the reagents from reagent containers to the microfluidic valves 10 via transport lines. Therefore, the microfluidic valves 10 for the fluidic connections 11 of the reagents required for the synthesis are opened in sequence, one at a time, together with the microfluidic valve for a first outlet W1. Between the individual feeds, the main conduit channel 12 is first flushed in each case by simultaneously opening the microfluidic valve 10 for the solvent SOL and the microfluidic valve for the first outlet W1, and then dried by opening the microfluidic valve for the inert gas GAS and the microfluidic valve for the first outlet W1. As a result, none of the reagents enters the synthesis chamber 13 during the feeding, rinsing and drying processes. By simultaneously opening the microfluidic valve for the inert gas GAS and one of the microfluidic valves 10 for each of the fluidic connections 11 of the reagents required for the synthesis, the reagents can be conveyed back to the reagent containers after the synthesis has ended.

[0066] In the following, the synthesis steps of a synthesis cycle will be discussed, which are necessary for coupling a nucleotide to the end of a partial sequence of an oligonucleotide or as the first nucleotide to a linker molecule of a carrier medium. The sequence of the synthesis steps and the reagents used for them are known per se.

[0067] Each oligonucleotide starts at a linker molecule of a carrier medium located in the synthesis chamber 13 and is extended with each synthesis step by one nucleotide, which is coupled to the end of the chain. The 5′-OH group of the oligonucleotide is provided with an acid-labile dimethoxytrityl protecting group (4,4′-dimethoxytrityl—DMT).

[0068] First, a reagent for detritylation DEBL of one end of an oligonucleotide containing a partial sequence or for detritylation of the linker molecule is supplied to the synthesis chamber 13 from the corresponding reagent container. In this process, the microfluidic valve 10 controlling the fluidic connection 11 for the reagent for detritylation DEBL is opened together with the microfluidic valve controlling the second outlet W2, as described above.

[0069] This removes the DMT protecting group so that another nucleotide can be coupled to the free 5′-OH group. In the present case, the reagent for detritylation DEBL is an acidic solution, namely a solution containing 2% trichloroacetic acid or 3% dichloroacetic acid in an inert solvent such as dichloromethane or toluene. This step is also referred to as the deblocking step.

[0070] In the next step, the nucleotide chain is extended at the detritylated free 5′-OH group by one base, i.e. either adenine B1, guanine B2, cytosine B3 or thymine B4 for a DNA strand or uracil B4 for an RNA strand. For this purpose, a reagent for activating ACT of the free 5′-OH group and a reagent containing phosphoramidite of the corresponding base B1,B2,B3,B4 are alternately supplied to the synthesis chamber 14. The phosporamidites are fed dissolved in a solvent SOL, in particular in acetonitrile. In the present case, activation of the 5′-OH group is achieved by means of a 0.2-0.7 molar solution of an acidic azole catalyst, in particular by 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole or 4,5-dicyanoimidazole. In this process, the nucleotide couples to the free 5′-OH group of the oligonucleotide, while the phosphoramidite residue is cleaved off. The 5′-OH group of the newly coupled nucleotide is again protected by a DMT protecting group. This step is also referred to as the coupling step.

[0071] In the synthesis chamber 13, a mixture of two reagents for blocking CAP1,CAP2 unreacted 5′-OH groups is added in the next step. In the present case, blocking of unreacted 5′-OH groups is achieved by a mixture of acetic anhydride and 1-methylimidazole as catalyst. This step is also referred to as the capping step.

[0072] The final step in a synthesis cycle is the oxidation of a phosphite triester bond formed between the newly coupled nucleotide and the corresponding 5′-OH group of the oligonucleotide by adding a reagent for oxidation OXI. The reagent for oxidation OXI oxidizes the phosphite triester bond into a four-coordinated phosphotriester, a protected precursor of the naturally occurring phosphate diester internucleoside bond. This stabilizes the bond between the coupled nucleotide and the corresponding 5′-OH group. In the present case, oxidation is achieved under anhydrous conditions using (1S)-(+)-(10-camphersulfonyl)-oxaziridine (CSO). This step is referred to as the oxidation step.

[0073] The four steps of a synthesis cycle are repeated in the order of the nucleotide sequence of the oligonucleotide to be synthesized until the oligonucleotide has the predetermined length and sequence. The order of the nucleotide sequence can, for example, be predetermined by an appropriately automated device and can be set by a user, so that a plurality of different individually predeterminable oligonucleotides can be synthesized in a device with such a chip 14.

[0074] Once the oligonucleotide to be synthesized is completed, a reagent for cleaving the oligonucleotides from the linker molecules and for removing the protecting groups CL/DE is supplied to the synthesis chamber 13. In the present case, the reagent used for cleaving the oligonucleotides from the linker molecules and for removing the protecting groups CL/DE is a mixture of ammonia and methylamine, wherein the two reagents are preferably present in equal amounts in the mixture. This reagent CL/DE dissolves the oligonucleotides from the linker molecules, wherein this process takes about 3 to 15 minutes, usually about 5 minutes. During this process, the microfluidic valve controlling the fluidic connection 11 for a product collection vessel PRO is open to deliver the synthesized oligonucleotides into the product collection vessel PRO. Removal of the protecting groups typically takes an additional 3 to 15 minutes, typically about 5 minutes, when the product collection vessel PRO is brought to a temperature between 50° and 750, preferably about 65° C., for example via a built-in heating block. At room temperature, this process requires between 45 and 120 minutes, usually about 60 minutes.

[0075] After completion of the synthesis, the PRO product collection container can be removed and further processed.

[0076] It is understood that the described microfluidic valves 10 can be used in a variety of different microfluidic systems and microfluidic chips.

LIST OF REFERENCE SIGNS

[0077] 1 Inflow duct [0078] 2 Supply channel [0079] 3 Outflow channel [0080] 4 Membrane [0081] 5 Valve chamber [0082] 6 Connection channel [0083] 7 Carrier layer [0084] 8 Flexible membrane layer [0085] 9 Sealing edge [0086] 10 Microfluidic valve [0087] 11 Fluidic connection [0088] 12 Main conduit channel [0089] 13 Synthesis chamber [0090] 14 Microfluidic chip [0091] SOL Solvent [0092] GAS Inert gas [0093] ACT Reagent for the activation of a detritylated 5′-OH group. [0094] B1 Base 1 (e.g. phosphoramidite of the base adenine) [0095] B2 Base 2 (e.g. phosphoramidite of the base guanine) [0096] B3 Base 3 (e.g. phosphoramidite of the base cytosine) [0097] B4 Base 4 (e.g. phosphoramidite of the base thymine or uracil) [0098] W1 First outlet [0099] CL/DE Reagent to cleave the oligonucleotides from the linker molecules and/or a reagent to remove the protecting groups [0100] DEBL Reagent for the detritylation of a 5′-OH group provided with a dimethoxytrityl protecting group [0101] OXI Reagent for the oxidation of a phosphite triester bond [0102] CAP1 First reagent for blocking unreacted 5′-OH groups [0103] CAP2 Second reagent for blocking unreacted 5′-OH groups [0104] PRO Product collection container [0105] W2 Second outlet