Device for synthesizing oligonucleotides

Abstract

The invention relates to a device for synthesising oligonucleotides, comprising: a reagent container receptacle (1) for holding a reagent container support (17) comprising multiple reagent containers (18); an exchangeable microfluid chip (10) comprising a synthesis chamber, fluid connectors and microfluid valves; a control device (5); fluid connecting means (2); wherein the device can be loaded with the microfluid chip (10) and the reagent container support (17) when in a loading position; a chip receptacle (3). To allow cost-effective and prompt synthesis even of small amounts of oligonucleotides, the invention provides for an actuator device (6) to be provided, with which the reagent container receptacle (1), the microfluid chip (10) and the fluid connecting means (2) can be brought from the loading position to an operating position, in which operating position the reagent container receptacle (1), the chip receptacle (3) and the fluid connecting means (2) are positioned relative to each other such that reagents can be conveyed out of the reagent containers (18) towards the synthesis chamber (14) depending on the valve position of the microfluid valves.

Claims

1. A device for synthesizing oligonucleotides in which the device comprises: a reagent container receptacle for holding a reagent container support comprising multiple reagent containers for reagents for the synthesis of oligonucleotides; an interchangeable microfluid chip comprising a synthesis chamber for the synthesis of oligonucleotides, fluid connectors for the conveying of the reagents as well as microfluid valves, wherein the fluid connectors are connected to the synthesis chamber via a fluid connection and the fluid connection between a respective fluid connector and the synthesis chamber can be interrupted or released by means of a microfluid valve; a control device for controlling the microfluid valves; fluid connecting means for the transport of the reagents from the reagent containers to the fluid connectors of the microfluid chip, wherein in a loading position the device can be loaded with the microfluid chip, and the reagent container receptacle can be loaded with the reagent container support, a chip receptacle for holding the microfluid chip, wherein an actuator device is provided, with which the reagent container receptacle, the microfluid chip, by means of the chip receptacle, and the fluid connecting means can be brought from the loading position to an operating position, in which operating position the reagent container receptacle, the chip receptacle and the fluid connecting means are positioned relative to each other by means of the actuator device in such a way, that the microfluid valves of the microfluid chip are connected to the control device, that the reagent containers arranged in the reagent container receptacle are connected to the fluid connectors of the microfluid chip via the fluid connecting means, so that depending on the valve position of the microfluid valves reagents can be conveyed from the reagent containers in the direction of the synthesis chamber.

2. The device according to claim 1, wherein the control device has a pneumatic system for controlling the microfluid valves.

3. The device according to claim 1, wherein the actuator device has a linear movement axis and the positioning of the reagent container receptacle or fluid connector pieces or chip receptacle occurs along the linear movement axis.

4. The device according to claim 1, wherein the actuator device has a rack, wherein the fluid connecting means are attached firmly to the rack and wherein the reagent container receptacle and the chip receptacle are shiftably or swivellably attached to the rack in the direction of the fluid connecting means, in order for the reagent container receptacle and the microfluid chip to be moved from the loading position to the operating position and vice versa.

5. The device according to claim 1, wherein the chip receptacle has a connector portion for the connection of the microfluid chip to the control device, wherein the connector portion has control line channels for the pneumatic system of the control device, which are connected to a respective microfluid valve of the microfluid chip in the operating position.

6. The device according to claim 1, wherein the components of the device in the operating position are positioned relative to each other in the following order: reagent container receptacle fluid connecting means chip receptacle with microfluid chip, wherein the fluid connectors are arranged on a side of the microfluid chip facing the fluid connecting means and the microfluid valves are attached to the control device on a side of the microfluid chip opposite to the fluid connectors.

7. The device according to claim 1, wherein each fluid connecting means comprises a connector piece for connection with one of the fluid connector of the microfluid chip and a needle-like transport line for connection to one of the reagent containers and that pneumatic connecting means are provided, which are connectable to the reagent containers, via which pneumatic connecting means in the operating position a pneumatic system is connected to the reagent containers arranged in the reagent container receptacle, in order to convey reagents from the reagent containers via the fluid connecting means in the direction of the microfluid chip by means of the gas pressure of the pneumatic system.

8. The device according to claim 7, wherein, in the operating position, one end of a transport line of one of the fluid connecting means and one end of one of the pneumatic connecting means is respectively positioned within one of the liquid reagent containers, in order to convey reagents from the reagent container in the direction of the synthesis chamber by means of gas pressure of the pneumatic system, wherein the transport line and the pneumatic connecting means penetrate a sealing element of the corresponding reagent container.

9. The device according to claim 7, wherein, in the operating position, at least one pair of corresponding reagent containers is provided in the reagent container receptacle, wherein one of the reagent containers contains a solid, namely, phosphoramidite of the bases adenine, guanine, cytosine, thymine or uracil in powder form, and the other one of the reagent containers contains a solvent, and that in the operating position one end of one of the pneumatic connecting means is arranged in the reagent container holding the solvent, and one end of one of the transport lines is arranged in the reagent container holding the solid, and that a connecting line connects both of the reagent containers with each other, so that via the gas pressure of the pneumatic system, solvents are transferred from the one reagent container into the other reagent container, the solid in the reagent container dissolves in the solvent and the solution can be conveyed in the direction of the synthesis chamber.

10. The device according to claim 7, wherein the control device has a pneumatic system for controlling the microfluid valves and that the reagent containers are connected via the pneumatic connecting means with the pneumatic system of the control device.

11. The device according to claim 7, wherein the fluid connecting means and the pneumatic connecting means are fixed to a support plate, said support plate being firmly attached to a rack in the actuator device, wherein the support plate has gas line channels, via which gas line channels the pneumatic system is connected to the pneumatic connecting means in the operating state.

12. The device according to claim 7, wherein the reagent containers are sealed by a respective sealing element, wherein the pneumatic connecting means and/or the fluid connecting means are configured in such a way that with the movement from the loading position to the operating position the sealing elements of a plurality of the reagent containers are penetrated simultaneously.

13. The device according to claim 1, wherein the reagent container support comprises at least six reagent containers and is arranged in the reagent container receptacle, wherein each of the reagent containers contains one of the following reagents: one reagent containing phosphoramidites of the bases adenine, guanine, cytosine, thymine or uracil; one reagent for the detritylation of an end of an oligonucleotide; one reagent for the activation of a detritylated 5′-OH group of an oligonucleotide and for the coupling of the phosphoramidite; one reagent for the oxidation of a phosphite triester; optionally at least one reagent for blocking non-implemented 5′-OH groups of activated oligonucleotides; wherein each reagent container is sealed with a sealing element.

14. The device according to claim 1, wherein the fluid connection of the microfluid chip comprises a main channel connected to the synthesis chamber and a plurality of fluid connecting channels, wherein each fluid connector is connected to the main channel via a fluid connecting channel and a microfluid valve.

15. The device according to claim 1, wherein the microfluid chip is configured either in one layer, wherein the microfluid valves are configured as membranes and arranged on an exterior surface of the microfluid chip or that the microfluid chip is configured with multiple layers, wherein the microfluid chip comprises a first support layer comprising the fluid connectors as well as a second support layer, wherein the second support layer is designed for connection to the control device and the microfluid valves are positioned between the first and the second support layer.

16. A method for operating a device for synthesizing oligonucleotides, the device comprising a reagent container receptacle in which a reagent container support comprising multiple reagent containers filled with reagents is arranged; a microfluid chip with a synthesis chamber for the synthesis of the oligonucleotide, with microfluid valves and with fluid connectors for the conveying of the reagents; a control device for the control of the microfluid valves; fluid connecting means for the transport of the reagents from the reagent containers to the fluid connectors of the microfluid chip, wherein the following steps are carried out: positioning of the microfluid chip in the device, so that the fluid connectors of the microfluid chip are aligned flush with the fluid connecting means and the microfluid valves of the microfluid chip can be connected to the control device; positioning of the microfluid chip by means of a chip receptacle that is moveable via an actuator device until a connection between the fluid connectors of the microfluid chip and the fluid connecting means is created; connection of the microfluid valves to the control device; positioning of the reagent container receptacle by means of the actuator device until a connection between the reagent containers and the fluid connectors of the microfluid chip is created via the fluid connecting means, so that depending on the valve position of the microfluid valves reagents can be conveyed out of the reagent containers in the direction of the synthesis chamber.

17. The method according to claim 16, wherein the control device includes a pneumatic system for the control of the microfluid valves and that the microfluid valves are connected to the pneumatic system of the control device.

18. The method according to claim 16, wherein the chip receptacle has a connector portion for the connection of the microfluid valves of the microfluid chip to the control device, in particular to the pneumatic system of the control device, wherein the connector portion is swiveled by means of the actuator device during the movement into the operating position, in order to connect the microfluid valves to the control device, wherein the microfluid chip is clamped between the fluid connecting means and the connector portion.

19. The method according to claim 16, wherein the reagent containers are connected to a pneumatic system via pneumatic connecting means and reagents are transported out of the reagent containers in the direction of the synthesis chamber via gas pressure of the pneumatic system.

20. The method according to claim 19, wherein the reagent containers are closed by a respective sealing element and the pneumatic connecting means and/or the fluid connecting means during positioning simultaneously penetrate the sealing elements of a plurality of the reagent containers.

21. The method according to claim 16, wherein before the start of the synthesis of an oligonucleotide the following steps are carried out: opening of a microfluid valve that controls a fluid connector for the conveying of reagents out of a reagent container; closing of the opened microfluid valve when reagent flows through the microfluid valve; washing of a main channel of the microfluid chip by opening a microfluid valve that controls a fluid connector for conveying a solvent; optionally drying of main channel of the microfluid chip by opening a microfluid valve that controls the conveying of an inert gas; repeating the above steps until each reagent from the reagent containers connected to the fluid connectors of the microfluid chip has been conveyed to the respective microfluid valve of the corresponding fluid connector.

22. The method according to claim 16, wherein for the synthesis of an oligonucleotide the following steps are carried out: opening of a microfluid valve that controls a fluid connector for the conveying of a reagent for the detritylation of one 5′-OH group of the oligonucleotide containing a dimethoxytrityl protecting group or for the detritylation of a linker molecule of a carrier medium from a reagent container; alternate opening of a microfluid valve, that controls a fluid connector for the conveying of a reagent for the activation of the detritylated 5′-OH group from a reagent container and of a microfluid valve, that controls a fluid connector for the conveying of a reagent containing phosphoramidites of the bases adenine or guanine or cytosine or thymine or uracil from a reagent container; optionally opening at least one microfluid valve that controls a fluid connector for the conveying of a reagent for the blocking of non-used 5′-OH groups from a reagent container; opening of a microfluid valve that controls a fluid connector for the conveying of a reagent for the oxidation of a phosphite triester bond from a reagent container; repeating of the previous steps.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The invention will now be explained in more detail with the help of embodiments. The drawings are examples and are intended to illustrate the idea behind the invention, but in no way restrict it or attempt to reproduce a definitive version.

(2) In particular:

(3) FIG. 1 shows a three dimensional view of a device according to the invention for the synthesis of oligonucleotides in a loading position;

(4) FIG. 2 shows a side view of the device in the loading position;

(5) FIG. 3 shows a side view of the device in an intermediate position

(6) FIG. 4 Side view of the device in an operating position

(7) FIG. 5 shows a frontal view of the device in the operating position;

(8) FIG. 6 shows a sectional view of the device according to line AA in FIG. 5;

(9) FIG. 7 shows an enlarged detailed view of the device according to area B in FIG. 6;

(10) FIG. 8 shows an enlarged detailed view of the device according to area C in FIG. 7;

(11) FIG. 9 shows an enlarged detailed view of the device according to area C in FIG. 7 in the intermediate position;

(12) FIG. 10 shows the layout of a first embodiment variant of a microfluid chip according to the invention;

(13) FIG. 11 shows an enlarged detailed view of a second embodiment variant of the device for a second embodiment variant of the microfluid chip analogous to FIG. 8;

(14) FIG. 12 shows an enlarged detailed view of the device in the intermediate position for a second embodiment variant of the microfluid chip analogous to FIG. 9;

(15) FIG. 13 shows the layout of a second embodiment variant of the microfluid chip according to the invention;

(16) FIG. 14 shows the layout of a embodiment variant of a reagent container support.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

(17) FIG. 1 shows a first embodiment variant of a device according to the invention for the synthesis of oligonucleotides in a loading position. In this embodiment the device comprises a reagent container receptacle 1, a chip receptacle 3 as well as a rack 8 and a support plate 9. Between the chip receptacle 3 and the reagent container receptacle 1 there are fluid connecting means 2 that are connected to the support plate 9.

(18) In the illustrated loading position, the reagent container receptacle 1 can be loaded with a reagent container support 17 that comprises multiple connected reagent containers 18 as a unit for the synthesis of the oligonucleotides. In the loading position illustrated, the reagent containers 18 are not permanently attached to the device, so that the reagent container support 17 is removable from the reagent container receptacle 1 or can be inserted into the reagent container receptacle 1 (see FIGS. 2 and 3). In other words, the reagent container support 17 is replaceable in the loading position by a similarly constructed reagent container support 17 acting as a spare part.

(19) In the illustrated loading position, the chip receptacle 3 can be loaded with a microfluid chip 10 made of plastics material, which microfluid chip 10 has a synthesis chamber 14, in which the synthesis of the oligonucleotides takes place (see FIGS. 10 and 13). As can be seen clearly from the figure, the microfluid chip 10 is not permanently attached to the device in the loading position, so that the microfluid chip 10 can be inserted in the device or can be removed from the device. In other words, the microfluid chip 10 is replaceable in the loading position by a similarly constructed microfluid chip 10 acting as a spare part. In this embodiment the chip receptacle 3 a drawer-like, removable receptacle compartment 3a, in which the microfluid chip 10 can be placed.

(20) As illustrated in particular in the FIGS. 7 to 13, the microfluid chip 10 has on the one side a plurality of fluid connectors 11. The fluid connectors 11 can be combined with the fluid connecting means 2, which fluid connecting means 2 in an operating position (see in particular FIGS. 4, 7 and 11) each connect a reagent container 18 with a fluid connector 11 of the microfluid chip 10, so that reagents are conveyable from the reagent containers 18 in the direction of the synthesis chamber 14. The fluid connecting means 2 are arranged between the chip receptacle 3 and the reagent container receptacle 1.

(21) In addition to the fluid connectors 11, the microfluid chip 10 has microfluid valves 12, which can be controlled via a schematically illustrated control device 5. The microfluid valves 12 of the microfluid chip 10 can be connected to the control device 5, wherein the microfluid valves 12 in the operating position are each connected to one control line 28 on the control device 5. In this embodiment the microfluid valves 12 on one of the fluid connectors 11 on the opposite side of the microfluid chip 10 are connectible to the control device 5.

(22) In order to be able to connect the microfluid valves 12 to the control device 5, the chip receptacle 3 has a connector portion 3b, which, in the operating position, connects the control device 5 to the microfluid chip 10.

(23) In order to bring the microfluid chip 10 together with the chip receptacle 3, the reagent container receptacle 1 and the fluid connecting means 2 from the loading position into the operating position, in this embodiment of the device an actuator device 6 is provided, which comprises the rack 8 and a track 8a fixed to the rack 8. The support plate 9 is fixed firmly to the rack 8 in this, while the chip receptacle 3 and the reagent container receptacle 1 are fixed in a translationally moveable manner along track 8a. Each of the fluid connecting means 2 comprises one connector piece 2a for connection to the fluid connectors 11 of the microfluid chip 10 and a needle-like transport line 2b for the transport of the reagents from the reagent container 18. The actuator device 6 comprises preferably at least one electrical power unit, for example an electric motor or a linear motor and or a spindle drive.

(24) The FIGS. 2 to 4 show the motion sequence of the device from the loading position to the operating position.

(25) In FIG. 2 the receptacle compartment 3a is extended and the microfluid chip 10 is placed in the receptacle compartment 3a. The receptacle compartment 3a is opened in the direction of the fluid connecting means 2, whereby the microfluid chip 10 is lying in a peripheral area in the receptacle compartment 3a. Thereby the microfluid chip 10 can preferably only be inserted in one direction in the receptacle compartment 3a and is fixed in position in receptacle compartment 3a. The chip receptacle 3 has a connecting portion 3c, with which the chip receptacle 3 is connected to the track 8a. Furthermore it is illustrated how a reagent container support 17 is positioned in the reagent container receptacle 1. Thereby it is advantageous if the reagent container receptacle 1 has positioning aids, such as positioning lugs, tracks or stops, in order to ensure that the reagent container support 17 can only be inserted in one direction in the reagent container receptacle 1 or that the reagent container support 17 is separable simply and easily in a predefined relative position with respect to the fluid connecting means 2 applied. The reagent container receptacle 1 also has a connecting portion 1a with which the reagent container receptacle 1 is connected to the track 8a.

(26) The actuator device 6 facilitates the positioning of the chip receptacle 3, the reagent container receptacle 1 and the fluid connecting means 2 along a linear movement axis 7. In this embodiment this is achieved by aligning the track 8a parallel to the movement axis 7.

(27) In this embodiment the conveying of the reagents is achieved via gas pressure that originates from a pneumatic system. The gas in the pneumatic system is an inert gas, wherein in this embodiment argon is used. In order to connect the reagent container 18 with the pneumatic system, pneumatic connecting means 4 are provided, which are each configured as a needle-like transport line. The pneumatic connecting means 4 are, preferably running parallel to the fluid connecting means 2, firmly secured to the support plate 9.

(28) In FIG. 3 an intermediate position is shown in which the drawer-like receptacle compartment 3a is pushed closed in the chip receptacle 3, wherein the insertion direction is normal to the linear movement axis 7. Thereby the fluid connectors 11 of the microfluid chip 10 inserted in the receptacle compartment 3a are aligned to the fluid connecting means 2, more precisely to their connector pieces 2a, so that in the case of a shift in the chip receptacle 3 along the movement axis 7 in the direction of the support plate 9 a connection between the fluid connectors 11 and the fluid connecting means 2, more precisely to their connector pieces 2a, can be created.

(29) The reagent container support 17 is positioned in the reagent container receptacle 1 in such a way that the individual reagent containers 18 are aligned on the one hand to the fluid connecting means 2, in particular to their transport lines 2b, and on the other hand are aligned to the pneumatic connecting means 4 configured as transport lines, so that in the case of a shift in the reagent container receptacle 1 along the movement axis 7 in the direction of the support plate 9 a connection between the connecting means 2, more precisely to their transport lines 2b, and the reagent containers 18 as well as between the pneumatic connecting means 4 and the reagent containers 18 can be created.

(30) If the positions of the reagent container receptacle 1 and the chip receptacle 3 in the figure is compared with that in FIG. 4, it can be seen that both the reagent container receptacle 1 as well as the chip receptacle 3 have already been pushed in the direction of support plate 9.

(31) FIG. 4 now shows the operating position of the device in which the fluid connectors 11 of the microfluid chip 10 are connected to the connector pieces 2a of the fluid connecting means 2 and one end of a respective transport line 2b of a fluid connecting means 2 and one end of a pneumatic connecting means 4 are found within a reagent container 18 (see in particular FIGS. 6 and 7). The reagent container receptacle 1 and the chip receptacle 3 run along the track 8a in the direction of the linear movement axis 7 in a maximum position.

(32) In FIG. 5 the frontal view of the device is illustrated, which shows in particular, that the reagent container support 17 has several rows of reagent containers 18. Thereby the fluid connecting means 2 and the pneumatic connecting means 4 are positioned behind each other in the selected view.

(33) FIG. 6 now shows a sectional view of the device in the operating position, wherein on the one hand the connection of the microfluid chip 10 to the fluid connecting means 2 as well as to the control device 5 are visible and on the other hand the connection of the pneumatic 4 and the fluid connecting means 2 with the reagent containers 18.

(34) While the connection of the microfluid chip 10 based on the FIGS. 8 and 9 have been explained in detail, the mechanism for the transport of the reagents in the direction of the microfluid chip 10 shall be discussed based on FIG. 7.

(35) The support plate 9 has a plurality of gas line channels 21, through which the pneumatic connecting means 4 are connected to the gas lines 27 of the pneumatic system. In the present embodiment the pneumatic connecting means 4 are configured as transport lines, which are stuck onto or pressed into openings in the support plate 9. In order to control the conveying of the reagents, the gas line channels 21 are controllable via at least one conveying valve, which can release or block the connection of the gas line channels 21 with the gas lines 27 of the pneumatic system. Preferably for each pneumatic connecting means 4 or for each gas line 27 a dedicated conveying valve is provided.

(36) Only fluid reagents can be conveyed in the direction of the microfluid chip 10 via the fluid connecting means 2. The reagent container 18 illustrated to the far right of the figure contains a fluid reagent. As illustrated, the end of the transport line 2b of the fluid connecting means 2 facing away from the microfluid chip 10 is positioned in the reagent container 18. Thereby the transport line 2b of the fluid connecting means 2 penetrates a sealing element 19 of the reagent container 18 during the positioning of the reagent container receptacle 1. The end of the transport line 2b of the fluid connecting means 2 is positioned in this in the bottom area of the reagent container 18 and thus in other words is immersed in the reagent. The connector piece 2a of the fluid connecting means 2 is attached to the side of the support plate 9 facing away from the reagent container 18, so that the transport line 2b of the fluid connecting means 2 pass through the support plate 9.

(37) In analogy to the fluid connecting means 2 the end of the pneumatic connecting means 4 configured as a transport line facing opposite to the support plate 9 is positioned in the reagent container 18. Thereby a needle-like end of the pneumatic connecting means 4 also penetrates the sealing element 19 of the reagent container 18 during the positioning of the reagent container receptacle 1. Thereby, the end of the pneumatic connecting means 4 is positioned in an area of the reagent container 18 facing opposite to the bottom area.

(38) Through the sealing element 19 a closed volume is formed within the reagent container 18. When the gas pressure in the reagent container 18 is increased by the gas supply via the pneumatic connecting means 4, the liquid reagent is conveyed in the direction of the microfluid chip 10 via the transport line 2b of the fluid connecting means 2.

(39) Certain reagents cannot be kept in liquid form for a long time since they lose their reactivity. In this case a pair of reagent containers 18 are provided, wherein one of the reagent containers 18 contains a solvent SOL1, SOL2, SOL3, SOL4 and the other reagent container 18 a solid to be dissolved. In this exemplary embodiment the solvent is acetonitrile and the solid is phosphoramidite of the bases adenine B1, guanine B2, cytosine B3, thymine or uracil B4.

(40) The fluid connecting means 2 are configured in analogy to the fluid connecting means 2 described above, wherein the end of the transport line 2b of the fluid connecting means 2 is arranged in the reagent container 18 holding the phosphoramidite B1, B2, B3, B4 in powder form. The pneumatic connecting means 4 are also configured in analogy to the pneumatic connecting means 4 described above, however the end of the pneumatic connecting means 4 is arranged in the reagent container 18 holding the solvent SOL1, SOL2, SOL3, SOL4. The reagent container 18 holding the phosphoramidite B1, B2, B3, B4 in powder form is connected to the reagent container 18 holding the solvent SOL1, SOL2, SOL3, SOL4 by one of the fixed connecting lines 20 in the support plate 9. When the gas pressure in the reagent container 18 holding the solvent SOL1, SOL2, SOL3, SOL4 is increased by conveying of gas via the pneumatic connecting means 4, the solvent SOL1, SOL2, SOL3, SOL4 is transferred to the reagent container 18 holding the phosphoramidite B1, B2, B3, B4 in powder form via the connecting line 20 where the phosphoramidite B1, B2, B3, B4 in powder form is dissolved in the solvent SOL1, SOL2, SOL3, SOL4. As soon as all of the solvent SOL1, SOL2, SOL3, SOL4 has been transferred, gas flows over the connecting line 20 in the reagent container 18 now containing the solution, so that via the gas pressure the now solution can be conveyed in the direction of the microfluid chip 10 via the fluid connecting means 2.

(41) In alternative embodiment variants it can also be provided that the solvent SOL is conveyed from a central solvent container into the reagent container 18 holding the phosphoramidite B1, B2, B3, B4 in powder form and not via four separate reagent containers 18 holding the solvent SOL1, SOL2, SOL3, SOL4.

(42) Fundamentally, for the control of the microfluid valves 12 differently configured control devices 5 are conceivable, for example piezoelectrically driven stoppers or lids. In this exemplary embodiment the actuation of the microfluid valves 12 is carried out via a pneumatic system of the control device 5. While it is generally conceivable to provide for two separate pneumatic systems, one for the conveying of the reagents and one for the actuation of the microfluid valves 12, it is advantageous if the device comprises a single pneumatic system. In this exemplary embodiment, a pneumatic system, i.e. the pneumatic system of the control device 5, is therefore present, so that the pneumatic system of the control device 5 is also connected to the gas line channels 21 in the support plate 9 via the gas lines 27. In the following the pneumatic system is further discussed for the sake of transparency.

(43) The pneumatic system is attachable via the connector portion 3b of the chip receptacle 3 to the microfluid valve 12 of the microfluid chip 10. For this purpose, the connector portion 3b has a control line channel 22 for each of the microfluid valves 12. Each control line channel 22 is thereby connected to one of the control lines 28 of the pneumatic system of the control device 5. For example, the control lines 28 can be pressed or glued in the control line channels 22. The connector portion 3b can for example be configured directly as part of the chip receptacle 3 or for example as a separate plate fastened to the chip receptacle 3.

(44) In order to be able to control the opening and closing of the microfluid valves 12 via the control device 5, each of the control lines 28 of the control device 5 is controllable via a separate control valve. Thereby the microfluid valves 12 in this exemplary embodiment are closed when the relevant control valve is open and vice versa.

(45) On the basis of FIG. 9, the connection of the microfluid chip 10 to the fluid connecting means 2 and to the control device 5 is described in the following. FIG. 9 shows a device still in an intermediate position, wherein the microfluid chip 10 is on a positioning edge of the receptacle compartment 3a and the fluid connectors 11 are configured flush to the connector pieces 2a of the fluid connecting means 2, however they are still spaced apart. The fluid connectors 11 in this exemplary embodiment are configured as openings, for example drill holes, in the underside of the microfluid chip 10. The connector pieces 2a are attached to the support plate 9, wherein between the connector pieces 2a a support element 24 is arranged, to prevent a deflection of the microfluid chip 10 in the operating position.

(46) The microfluid chip 10 has a first support layer 10a that forms the fluid connectors 11. These are thereby configured in this exemplary embodiment as openings in the underside of the microfluid chip 10. The connector pieces 2a of the fluid connecting means 2 are pressed onto the fluid connectors 11 and serve as further sealing elements that are pressed against the first support layer 10a or the underside of the chip 10 in the operating position, in order to guarantee the necessary impermeability of the connection with the reagent container 18. The fluid connectors 11 are oriented flush to the fluid connecting means 2.

(47) The microfluid chip 10 is configured on a multilayer level and has two support layers 10b, which are pressed against the sealing elements 23 in the operating position and form the upper surface of the microfluid chip 10. The microfluid valves in this exemplary embodiment are shifted to the side opposite the control line channels 22 and arranged between the two support layers 10a, 10b. Thereby the microfluid valves 12 in this exemplary embodiment are configured as membranes.

(48) In order to be able to actuate the microfluid valves 12 arranged within the microfluid chip 10 via the control lines 28 and the control line channels 22, the second support layer 10b has pneumatic connectors 15 configured as openings that are oriented flush to the control line channels 22. The microfluid valves 12 are connected to the pneumatic connectors 15 via pneumatic connecting channels 16. The actuation of a microfluid valve 12 is therefore carried out by the control device 5 via a control valve, a control line 28, a control line channel 22, a pneumatic connector 15 and a pneumatic connecting channel 16.

(49) The connector portion 3b has sealing elements 23 on one of the surfaces facing the microfluid chip 10 or the second support layer 10b, which guarantee the pneumatic connection between the control line channel 22 and the microfluid valve 12 in the operating position, wherein the transition between the control line channels and the connectors 15 are sealed.

(50) If the chip receptacle 3 is moved further in the direction of the support plate 9, the connector pieces 2a of the fluid connecting means 2 are clamped against the fluid connectors 11 or the first support layer 10a, wherein the microfluid chip 10 is lifted from the receptacle compartment 3a and clamped against a clamping surface of the connector portion 3b or against the sealing element 23. This operating position is shown in FIG. 8: The microfluid chip 10 is clamped between the clamping surface of the connector portion 3b or between the sealing elements 23 arranged on the clamping surface and the connector pieces 2a of the fluid connecting means 2. By clamping the connector pieces 2a to the fluid connectors 11 the connection between the reagent containers 18 and the microfluid chip 10 is sealed. The connector pieces 2a of the fluid connecting means 2 thereby also serve as further sealing elements and are preferably configured as malleable. The deformation of the sealing elements 23 by the clamping pressure guarantees the necessary impermeability of the connection between the control line channels 22 and the pneumatic connectors 15 of the microfluid chip 10 for the actuation of the microfluid valves. The support element 24 lies on the underside of the microfluid chip 10 formed by the first support layer 10a and prevents the deformation of the chip 10 due to the clamping.

(51) In order to be able to remove the microfluid chip 10 from the device after synthesis, the chip receptacle 3 is moved away from the support plate 9, in order to arrive at the configuration illustrated in FIG. 9.

(52) FIG. 10 shows a possible layout of a first embodiment variant of the microfluid chip 10, with which a synthesis of oligonucleotides is possible. The microfluid chip 10 has a fluid connection 13, which connects the synthesis chamber 14 with the fluid connectors 11. Each fluid connection 13 is blockable or releasable via a microfluid valve 12, so that via the position of the corresponding microfluid valve 12 the conveying of a reagent from one of the fluid connectors 11 to the synthesis chamber 14 can be achieved or blocked.

(53) The fluid connection 13 is divided into a main channel 13a via which the reagents flow in the direction of the synthesis chamber and a plurality of connection channels 13b, which connect the main channel 13a with one of the fluid connectors 11 respectively.

(54) Due to the offset of the microfluid valves 12 and the pneumatic connectors 15, a pneumatic connecting channel 16 connecting the respective valves 12 with the pneumatic connectors 15 is provided. The connecting channels 13b of the fluid connection 13 each end in a microfluid valve 12, so that the respective microfluid valve 12 is arranged between the main channel 13a and the connecting channel 13b.

(55) In the present embodiment variant the main channel 13a is divided into two portions extending parallel to each other, referred to as branches in the following, which join together in front of the synthesis chamber 14 in a synthesis channel 26. In the synthesis chamber 14 there is a carrier medium with linker molecules, which serves as the starting point for the synthesis of the oligonucleotides. The synthesis chamber 14 can be configured for example as a funnel shape and can widen out in the flow direction. In order not to flush out the carrier medium during the flow through the synthesis chamber 14, a retention structure 25 is arranged in the direction of the flow, preferably directly behind the synthesis chamber 14.

(56) In order to optimally exploit the construction space, one branch of the main channel 13a, at the end of which the fluid connector 11 for the solvent SOL and the pneumatic connector 15 for the inert gas GAS are arranged, two portions running parallel to each other are joined together on the one, preferably U-shaped, connecting piece. Thereby the connecting channels 13b of the reagents B1, B2, B3, B4 flow into the portion of the corresponding branch of the main channel 13a bordering the synthesis channel 26.

(57) How the flow through the fluid connection 13 of the microfluid chip 10 is achieved, is described in the following based on the solvent SOL. This principle can be used analogously for all the other reagents.

(58) In order to allow the solvent SOL to flow from the corresponding fluid connector 11 via the connecting channel 13b and the main channel 13a in the direction of the synthesis chamber 14, on the one hand the microfluid valve 12 controlling the connector 11 is opened. On the other hand a microfluid valve 12 is opened, which controls a further fluid outlet connector 11b configured as a second outlet W2. The second outlet connector 11b is arranged in the direction of the flow behind the synthesis chamber 14, so that the solvent SOL from the fluid connector 11 flows over the fluid connection 13, the synthesis chamber 14 and the second outlet connector 11b to the second outlet W2. The second outlet W2 can for example be configured through a waste container.

(59) The microfluid valves 12 are fundamentally kept in a closed position, so that the connecting channels 13b are blocked off from the main channel 13a or the fluid connection 13 is interrupted. The closed position is achieved by the control device 5 exercising pressure over the microfluid valves 12 via the pneumatic system, more precisely via the control lines 28, the control line channels 22, the pneumatic connectors 15. If the pressure exercised by the control device 5 on one of the microfluid valves 12 is reduced or the pressurization is suspended, the corresponding microfluid valve 12 opens and the fluid connection 13 between the corresponding connecting channel 13b and the main channel 13a is created or open.

(60) The connector 11 for the solvent SOL is attached in such a way to one side of a branch of the main channel 13a that none of the connecting channels 13b of the reagents necessary for the synthesis is positioned after this. Thus the fluid connection 13 can be washed by means of the solvent SOL, in order to remove the reagents. In order to be able to wash the second branch of the main channel 13a as well, a first outlet connector 11 a configured as outlet W1 is provided, which is attached at one side to the second branch of the main channel 13a. If the microfluid valves 12 controlling the first outlet connector 11a and the microfluid valves 12 controlling connector 11 for the solvent SOL are opened simultaneously, the solvent SOL flows over the first branch of the main channel 13a into the second branch of the main channel 13a, without flowing through the synthesis chamber 14.

(61) In addition to this a pneumatic connector 15 for an inert gas GAS is provided, which in this exemplary embodiment is connected to the pneumatic system. The pneumatic system contains the inert gas GAS, wherein this is argon in this exemplary embodiment. By the flow through the fluid connection 13 of the inert gas GAS, the fluid connection 13 is dried after washing with the solvent SOL.

(62) Before the synthesis can begin, it is necessary to convey the reagents from the reagent containers 18 via the transport lines 2b of the fluid connecting means 2 to the microfluid valves 12. Therefore, in sequence, the microfluid valves 12 for the fluid connectors 11, the reagents necessary for the synthesis one after the other together with the microfluid valves 12 for the first outlet connector 11a of the first outlet W1 are opened. Between the individual conveying the main channel 13a is first washed by the simultaneous opening of the microfluid valve 12 for the solvent SOL and the microfluid valve 12 for the first outlet connector 11a of the first outlet W1 and then dried by opening the microfluid valve 12 for the inert gas GAS and the microfluid valve 12 for the first outlet connector 11a of the first outlet W1. Thereby none of the reagents enter the synthesis chamber 14 during the conveying, the washing and the drying. Through the simultaneous opening of the microfluid valves 12 for the inert gas GAS and one of the microfluid valves 12 for the fluid connectors 11 of the reagents necessary for the synthesis respectively, the reagents can be conveyed back into the reagent container 18 after the end of the synthesis.

(63) In the following, the synthesis steps which are necessary in a synthesis cycle for the coupling of a nucleotide to the end of a subsequence of a oligonucleotide or as the first nucleotide to a linker molecule of a carrier medium are discussed. The sequence of the synthesis steps and the reagents used for this are known per se.

(64) Each oligonucleotide starts at a linker molecule of a carrier medium arranged in the synthesis chamber 14 and is increased by one nucleotide with each synthesis step, which is coupled to the end of the chain. The 5′-OH group of the oligonucleotide is furnished with an acid-labile dimethoxytrityl protecting group (4,4′-dimethoxytrityl—DMT).

(65) First of all, a reagent for the detritylation DEBL of one end of an oligonucleotide comprising a subsequence or for the detritylation of the linker molecule is conveyed to the synthesis chamber 14 from the corresponding reagent container 18. Thereby, as described previously, the microfluid valve 12 controlling the fluid connection 11 for the reagent for the detritylation DEBL is opened simultaneously with the second microfluid valve 12 controlling the outlet connector 11b.

(66) In this way the DMT protecting group is removed so that a further nucleotide can be couple to the 5′-OH group. In the case of the reagent for the detritylation DEBL, in the present case this an acidic solution, namely a solution containing 2% trichloroacetic acid or 3% dichloroacetic acid in an inert solvent such as acetonitrile, dichloromethane or toluol is conceivable. This step is also designated a deblocking step.

(67) In the next step the nucleotide chain on the detritylated, free 5′-OH group, is extended by an alkali, 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 a reagent for the activation of the free 5′-OH group and a reagent containing phosphoramidites with the corresponding base B1, B2, B3, B4 is alternately conveyed to the synthesis chamber. The phosphoramidites are conveyed dissolved in a solvent SOL, in particular acetonitrile. The activation of the 5′-OH group is in this example achieved by means of a 0.2-0.7 molar solution of an acid azole catalyst, in particular through 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthio tetrazole or 4,5-dicyanoimidazole. Thereby the nucleotide couples with the free 5′-OH group of the oligonucleotide, while the phosphoramidite residue is split. The 5′-OH group of the newly coupled nucleotide is once again protected by a DMT protecting group. This step is also called the coupling step.

(68) In the synthesis chamber 14 in the next step a mixture of two reagents is conveyed to the blocking CAP1, CAP2 of the non-used 5′-OH groups. In the present case the blocking of the non-used 5′-OH groups is achieved by a mixture of acetate anhydride and 1-methylimidazole as catalyst. This step is also called the capping step.

(69) In the last step in a synthesis cycle the oxidation of a phosphite triester coupling that is formed from the newly coupled nucleotide and the corresponding 5′-OH group of the oligonucleotide is carried out, in that a reagent is conveyed to oxidation OXI. The reagent for oxidation OXI oxidizes the phosphite triester bond in a fourfold coordinated phosphite triester, a protected forerunner of the naturally occurring phosphate diester internucleotide linkage. Thereby the bond between the coupled nucleotide and the corresponding 5′-OH group is stabilized. In the present case, oxidation is carried out under anhydrous conditions by means of (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO) or tert-butyl hydroperoxide (TBHP). This step is called the oxidation step.

(70) In the method according to the invention the microfluid chip 10 or the fluid connection 13 is washed and dried after each step in the synthesis cycle, as described above. It is particularly advantageous thereby if during the washing and drying the microfluid valves 12, the fluid outlet connectors 11a, 11b configured as outlet W1, W2 are alternately opened and closed.

(71) The opening duration of the microfluid valves 12, by means of which the quantities of reagents entering the synthesis chamber 14 and the duration of the synthesis step or the duration of the washing or drying are determined, is controlled by the control device 5. In particular, the control device 5 has a programming, which is configured in such a way that for each of the synthesis steps, the washing and drying processes, the necessary opening time of the corresponding microfluid 12 is determined.

(72) The four steps of a synthesis cycle are repeated in the order of the nucleotide sequence of the oligonucleotide to be synthesized as often as required until the oligonucleotide has the predetermined length and sequence. As soon as the oligonucleotide to be synthesized is ready, a reagent for the splitting of the oligonucleotides from the linker molecule and for the removal of the protecting groups CL/DE is conveyed to the synthesis chamber 14. In the present case, a mix of ammonia and methylamine is used as a reagent for the splitting of the oligonucleotides from the linker molecule and for the removal of the protecting groups CL/DE, wherein both reagents are preferably contained in the mixture in equal parts. This reagent CL/DE releases the oligonucleotide from the linker molecule, wherein this process lasts approximately 3 to 15 minutes, as a rule approximately 5 minutes. In particular, the fluid connector 11 is opened for a microfluid valve 12 controlling a product collection container PRO, in order to convey the synthesized oligonucleotide into the product collection container. The removal of the protective group lasts approximately a further 3 to 15 minutes, as a rule approximately 5 minutes, if the product collection container PRO, is brought to a temperature of between 50° and 75° , preferably 75° C., via an integral heating block. At room temperature this process requires between 45 and 120 minutes, as a rule approximately 60 minutes.

(73) After the conclusion of the synthesis, the product collection container PRO can be removed and further processed. The device can then be moved from the loading position to the operating position, wherein the previously described steps (FIGS. 4, 3, 2) are carried out in reverse order, in order to be able to remove or replace both the microfluid chip 10 as well as the reagent container support 17 from the device.

(74) FIGS. 11 and 12 show a second embodiment variant of the device in which a second, preferred embodiment variant of the microfluid chip 10 is used. Since the fundamental construction of the device remains unchanged, in the following simply the differences will be described. In particular, FIGS. 11 and 12 clarify in comparison to FIGS. 8 and 9, the structural differences of both of the embodiment variants. In both of the embodiment variants the pneumatic connecting means 4 are configured as transport channels directly connected to the support plate 9, which are bonded into the gas line channels 21, for example.

(75) The different structure of the microfluid chip 10 and the resulting differences regarding the connection to the fluid connecting means 2 and with the connection to the control device 5 are discussed based on FIGS. 11 and 12.

(76) In the second embodiment variant the microfluid chip 10 is configured in one layer, wherein preferably the microfluid valves 12 are also designed in the material of the microfluid chip 10, the microfluid chip 10 is therefore configured monlithically. The microfluid valves 12 are thereby designed on one side of the microfluid chip 10, in the present exemplary embodiment on the upper surface, and the fluid connectors 11 on the side facing the microfluid valves 12, in the present exemplary embodiment on the lower surface. The microfluid valves 12 face the connector portion 3b of the chip receptacle 3 and are aligned flush with the control line channels 22, however at a distance from these.

(77) In this embodiment variant, the microfluid valves 12 are connected directly to the control lines 28 or the control line channels 22, so that no pneumatic connectors 15 or no pneumatic connecting channels 16 are necessary. When the microfluid chip 10 is clamped between the connector pieces 2a of the fluid connecting means 2 and the sealing elements 23, as can be seen in FIG. 11, the sealing elements 23 seal the space between the microfluid valves 12 arranged on the upper side of the microfluid chip 10. By pressurization of the microfluid valves 12 via the control lines 28 of the pneumatic system, the microfluid valves 12 configured as a membrane are deformed and interrupt the fluid connection 13.

(78) Finally, FIG. 13 shows the layout of the second embodiment variant of the microfluid chip 10. As previously, only the differences to the layout described above will be discussed in the following. For the sake of transparency let it be said that the steps in the synthesis cycle are carried out completely analogously.

(79) In the present exemplary embodiment some of the microfluid valves 12 are arranged within the connecting channels 13b and not on one side in the transition between the connecting channels 13b and the main channel 13a. As mentioned earlier, in this embodiment variant neither pneumatic connectors 15 nor pneumatic connecting lines are envisaged in the microfluid chip 12.

(80) The inert gas GAS is conveyable via a fluid connector 11, which however, for example via the gas line channels 21, is attached to the pneumatic system. In alternative embodiment variants, two separate fluid connectors 11 or two separate reagent containers 18 can be envisaged for the reagent for the splitting of the oligonucleotides from the linker molecule and for the removal of the protecting groups CL/DE.

(81) In both of the embodiment variants of the microfluid chip 10, the fluid connectors 11 for the reagents for carrying out the synthesis cycles are connected to the main channel 13a in such a way that the fluid connectors 11 for the bases B1, B2, B3, B4 and the reagent for the activation ACT are connected to the first branch of the main channel 13a and the fluid connectors 11 for the reagent for the detritylation DEB, the reagent for the oxidation OXI as well as both of the reagents for the blocking of the non-used 5′-OH groups CAP1, CAP2 are connected to the second branch of the main channel 13a. It is self-evident that the bases B1, B2, B3, B4 can be attached to the main channel 13a in any preferred sequence.

(82) At the end of the first branch of the main channel 13a, the connectors 11, 15 for the conveying of the solvent SOL and the inert gas GAS are connected. At the end of the second branch of the main channel 13a, the first outlet connector 11a for the first outlet WI is connected. The second branch of the main channel 13a is furthermore connected to the fluid connector 11 for the reagent for the splitting of the oligonucleotides from the linker molecule and for the removal of the protecting groups CL/DE.

(83) At the transition between the two branches of the main channel 13a and the synthesis channel 26 there is also a microfluid valve 12 in the embodiment variant illustrated, which can close the synthesis channel 26, for example during the conveying of the reagents.

(84) In FIG. 14 the sequence of the reagent containers 18 containing the reagents in the reagent container support 17 is given as an example, wherein the sequence of the reagent containers 18 is determined according to the second, preferred embodiment of the microfluid chip 10.

(85) The reagent containers 18 are divided into three columns in the figure, wherein for the sake of transparency only the upper reagent container 18 in a column is provided with a reference mark.

(86) The middle column contains from top to bottom reagent containers 18 for the following reagents: reagents for the activation ACT of a detritylated 5′-OH group,

(87) phosphoramidite of the first base adenine B1 in powder form,

(88) phosphoramidite of the second base guanine B2 in powder form,

(89) phosphoramidite of the third base cytosine B3 in powder form,

(90) phosphoramidite of the fourth base thymine or uracil B4 in powder form as well as the solvent SOL.

(91) The left column comprises contains from top to bottom four reagent containers 18 containing solvent SOL, preferably acetonitrile, wherein the first reagent container 18 contains solvent SOL1 for the first base B1, the second reagent container 18 contains solvent SOL2 for the second base B2, the third reagent container 18 contains solvent SOL3 for the third base B3, and the fourth reagent container 18 contains solvent SOL4 for the fourth base B4. Thereby the reagent containers 18 for the solvents SOL1, SOL2, SOL3, SOL4 are always arranged in pairs with the corresponding reagent containers 18 for the bases B1, B2, B3, B4.

(92) The right column includes from top to bottom reagent containers 18 for the following reagents: a reagent for the splitting of the oligonucleotides from the linker molecule and for the removal of the protecting groups CL/DE, reagent for the detritylation DEBL of one of the 5′-OH groups containing a dimethoxytrityl protecting group, reagent for the oxidation OXI of a phosphite triester bond, second reagent for blocking CAP2 non-used 5′-OH groups, first reagent for blocking CAP1 non-used 5′-OH groups.

(93) Thereby the reagent containers 18 in the middle column are connectible to the fluid connectors 11 of the first branch of the main channel 13a of the microfluid chip 10 and the reagent containers 18 in the right column are connectible to the fluid connectors 11 of the second branch of the main channel 13a of the microfluid chip 10.

LIST OF REFERENCE NUMERALS

(94) 1 reagent container receptacle

(95) 1a connecting portion

(96) 2 fluid connecting means

(97) 2a connector piece

(98) 2b transport line

(99) 3 chip receptacle

(100) 3a receptacle compartment

(101) 3b connector portion

(102) 3c connecting portion

(103) 4 pneumatic connecting means

(104) 5 control device

(105) 6 actuator device

(106) 7 linear movement axis of the actuator device

(107) 8 rack

(108) 8a track

(109) 9 support plate

(110) 10 microfluid chip

(111) 10a first support level

(112) 10b second support level

(113) 11 fluid connector

(114) 11a first outlet connector

(115) 11b second outlet connector

(116) 12 microfluid valve

(117) 13 fluid connection

(118) 13a main channel

(119) 13b connecting channel

(120) 14 synthesis chamber

(121) 15 pneumatic connector

(122) 16 pneumatic connecting channels

(123) 17 reagent container support

(124) 18 reagent container

(125) 19 sealing element

(126) 20 connecting lines

(127) 21 gas line channel

(128) 22 control line channel

(129) 23 sealing element

(130) 24 support element

(131) 25 retention structure

(132) 26 synthesis channel

(133) 27 gas lines

(134) 28 control line

(135) SOL solvent

(136) GAS inert gas

(137) ACT reagent for activating a detritylated 5′-OH group

(138) B1 Base 1 (for example phosphoramidite of the base adenine)

(139) B2 Base 2 (for example phosphoramidite of the base guanine)

(140) B3 Base 3 (for example phosphoramidite of the base cytosine)

(141) B4 Base 4 (for example phosphoramidite of the base thymine or uracil)

(142) W1 first outlet

(143) CL/DE reagent for the splitting of the oligonucleotides from the linker molecules or a reagent for the removal of the protecting groups

(144) DEBL reagent for the detritylation of a 5′-OH group equipped with a dimethoxytrityl protecting group

(145) OXI reagent for the oxidation of a phosphite triester bond

(146) CAP1 first reagent for the blocking of non-used 5′-OH groups

(147) CAP2 second reagent for the blocking of non-used 5′-OH groups

(148) PRO product collection container

(149) W2 second outlet

(150) SOL1 solvent for the dissolution of base 1

(151) SOL2 solvent for the dissolution of base 2

(152) SOL3 solvent for the dissolution of base 3

(153) SOL4 solvent for the dissolution of base 4