METHOD FOR INCREASING THE DOSING PRECISION OF MICROFLUIDIC PUMPS OR VALVES, AND WELDING APPARATUS AND TENSIONING APPARATUS FOR CARRYING OUT THE METHOD

Abstract

The invention relates to a method for increasing the dosing precision of microfluidic pumps and valves based on a flexible cover film/diaphragm and a valve trough, in which the surface of the diaphragm facing the valve trough is heated with a laser beam.

Claims

1. A method for increasing the dosing precision of microfluidic pumps 1, 2, 3 or valves which have a flexible diaphragm (4) and a valve body (8) with at least one valve trough (5, 6, 7), the flexible diaphragm (4) being attached to the valve body (8) in order to cover the valve trough (5, 6, 7), wherein the surface (9) of the diaphragm (4) facing the valve trough (5, 6, 7) is heated with a laser beam.

2. The method according to claim 1, wherein the diaphragm (4) is welded to the valve body (8) by means of the laser beam.

3. The method according to claim 1, wherein the diaphragm (4) or the valve body (8) is provided with a heat-activatable adhesive.

4. The method according to claim 1, wherein the laser beam is used to produce an attachment of the diaphragm (4) to the valve body (8) as a seam along the edge of the valve trough (5, 6, 7).

5. The method according to claim 1, wherein the surface of the diaphragm (4) facing the valve trough (5, 6, 7) is heated by radiation impinging on the diaphragm (4).

6. The method according to claim 5, wherein the radiation impinges on the surface (9) through the diaphragm (4).

7. The method according to claim 5, wherein the radiation impinges on the surface (9) through the valve body (8).

8. The method according to claim 1, wherein the surface (9) of the valve body (8) is polished before the attachment.

9. The method according to claim 1, wherein the surface (9) of the valve body (8) is plasma etched before the attachment.

10. The method according to claim 1, wherein the surface (9) of the valve body (8) is etched with an ion beam before the attachment.

11. The method according to claim 1, wherein the surface (9) of the valve body (8) is smoothed by a chemical modification before the attachment.

12. The method according to claim 1, wherein the surface (9) of the valve body (8) is hydrophilized hydrophilized before the attachment.

13. The method according to claim 1, wherein the surface (9) of the valve body (8) has a mean roughness value (Ra value) of less than 100 nm, preferably less than 50 nm and very preferably less than 20 nm, prior to the attachment around the valve trough (5, 6, 7).

14. The method according to claim 1, wherein the pump is designed to deliver liquids with flow rates between 0.01 μL/h and 1 ml/h, but particularly with flow rates between 0.01 and 100 μL/h and very particularly in the range of 0.1 to 80 μL/h.

15. The method according to claim 1, wherein the pump for the delivery of liquids operates with a pump volume per pump stroke between 5 nL/stroke and 1 μL/stroke, but particularly with a pump volume between 25 nL/stroke and 500 nL/stroke and very particularly in the range of 75 to 250 nL/stroke.

16. The method according to claim 1, wherein the inaccuracy of the guidance of the laser beam in the x-y direction is more than 0.05 micrometers and less than 1 micrometer, preferably less than 50 micrometers and very preferably less than 5 micrometers.

17. The method according to claim 1, wherein different polymers with different transmission ranges are used for the diaphragm (4) and the valve trough (5, 6, 7) and are welded with UV laser, visible laser beams or with infrared laser.

18. The method according to claim 1, wherein the wavelength range of the laser beam is between 0.1 and 1000 micrometers, preferably between 0.4 and 50 micrometers and very preferably between 0.78 and 3 micrometers.

19. The method according to claim 1, wherein the power of the laser beam is between 0.01 and 1000 watts, preferably between 0.1 and 100 watts and very preferably between 3 and 50 watts.

20. The method according to claim 1, wherein the attachment is performed over a line whose width is between 20 micrometers and 3 micrometers, preferably between 30 and 500 micrometers and particularly preferably between 50 and 300 micrometers.

21-22. (canceled)

Description

[0027] Embodiments are shown in the drawing and are described below. In the drawing

[0028] FIG. 1 shows a microfluidic pump with a plurality of valves and empty valve troughs,

[0029] FIG. 2 shows the pump shown in FIG. 1 with two filled valve troughs,

[0030] FIG. 3 shows the pump shown in FIG. 1 with a filled valve trough,

[0031] FIG. 4 shows a tensioning apparatus for applying a diaphragm,

[0032] FIG. 5 shows a side view of a welding apparatus,

[0033] FIG. 6 shows a plan view of the welding apparatus shown in FIG. 5,

[0034] FIG. 7 shows position of the adjusting screws on the welding apparatus shown in FIG. 5,

[0035] FIG. 8 shows the position of the adjusting screws shown in FIG. 7 in a plan view,

[0036] FIG. 9 shows the position of the force sensors and positioning pins on the welding apparatus shown in FIG. 5,

[0037] FIG. 10 shows a view of a glass vacuum chamber,

[0038] FIG. 11 shows a section through the vacuum chamber shown in FIG. 10,

[0039] FIG. 12 shows a valve contour without weld seam,

[0040] FIG. 13 shows a weld contour, and

[0041] FIG. 14 shows a valve contour with weld seam.

[0042] FIGS. 1 to 3 show a pumping sequence of a plurality of microfluidic pumps 1, 2, 3, in which a flexible diaphragm 4 covers valve troughs 5, 6, 7 of a valve body 8. To attach the flexible diaphragm 4 to the valve body 8, the surface 9 of the diaphragm 4 facing the valve troughs 5, 6, 7 was heated with a laser beam. Since in the present case a plurality of valve troughs 5, 6, 7 are located next to each other, the diaphragm 4 is only attached to the valve body 8 in the edge area 10.

[0043] In the embodiment, the valve body 8 is a microfluidic chip 11, above which a microtiter plate 12 is arranged. This microtiter plate 12 contains reservoirs 13 and wells 14. The microtiter plate 12 is moved by a shaking array 15, in which channels 16, 17, 18 of a pneumatic system acting on the diaphragm are arranged.

[0044] FIG. 2 shows how liquid flows from the reservoir 13 into the valve troughs 5 and 6, and FIG. 3 shows how liquid in the valve trough 7 is connected to the well 14.

[0045] FIG. 4 shows a piston table 20 with a positioning table 21 arranged above it, over which a diaphragm 22 is tensioned. The diaphragm 22 rests on a polymer main body 23 and is held on both sides by magnets 24 and 25, which can be moved along a rail in the direction of the arrows 26, 27 to tension the diaphragm 22.

[0046] The complete system of the apparatus for welding a valve trough or pump trough and the cover film/cover diaphragm is shown in FIG. 5: the welding apparatus 30 contains a radiation source 31 (e.g. thulium fibre laser) and an axis system with the axes 32 and 33, which makes it possible to move the tensioning apparatus in a plane, but at least in one direction, under the laser in order to generate a weld seam with a defined position. The tensioning apparatus itself makes it possible to attach at least one valve trough, but normally two or more valve troughs or pump troughs integrated in a chip/polymer body 37 to the movable axis system. A transparent flexible diaphragm 38 is also tensioned over the polymer main body 37. The tensioning apparatus consists of a cylinder 39 with piston table 40, a positioning table 36, at least four adjusting screws 41 and at least four force sensors 42. The force sensors 42 allow the film to be tensioned isotropically with the aid of a tensioning collar 34. The tensioning apparatus with polymer main body and tensioned diaphragm is pressed against a glass plate 35 by lifting the piston table. The glass plate thus exerts a pressure on the polymer main body with chip. By introducing the energy of the laser through the glass plate onto the diaphragm tensioned over the polymer main body, both diaphragm and polymer main body are thermally softened or melted. The pressure between the glass plate and the polymer main body induces a material flow between the diaphragm and the polymer main body, which, after the solidification of the molten polymers, leads to a narrow, exactly positioned and mechanically very durable weld seam.

[0047] The polymer main body is precisely aligned on the positioning table by means of a centring apparatus consisting, for example, of 2 positioning pins 43, which fit into corresponding fitting holes on the main body, and is thereby brought into a fixed position (FIG. 9). The positioning table rests on four force sensors 42, which are embedded in the piston table, which is firmly connected to a cylinder. The four force sensors measure the forces applied to the four corners of the rectangular positioning table when the positioning table presses the polymer main body with the film tensioned over it against the glass plate 35 from below. The force distribution can be adjusted by means of four screws 41a, 41b, 41c, 41d at the corners (FIG. 7), which are fastened in the positioning table 36 via a thread. The screws reduce or increase the distance between the positioning table and the piston table so that the contact pressure at that point is reduced or increased, thus ensuring that the contact pressure is distributed evenly over the entire polymer main body.

[0048] The radiation source 31 is positioned at a distance with a certain focus position plane-parallel to the chip 37, so that the focus of the laser is either on or close to the plane spanned by the polymer main body and film. The closer the focus of the laser is to this plane, the narrower the weld seam becomes and the lower the beam power of the laser can be. The focus position also determines the energy input into the polymer body and the diaphragm film at the positions to be welded and thus the accuracy of the welding process. The focus position can either be fixed or variably adjusted via an axis system with the axes 32 and 33, which allows the laser to be moved perpendicularly to the arrangement of the polymer main body with film.

[0049] The polymer body 37 and the diaphragm 44 are pressed from below via the cylinder 39 against a glass plate 35, which has a high spectral transmission in the wavelength range of the laser. Especially in a wavelength range of 1940 nm, glass is highly suitable as a material for pressing the polymer body against the diaphragm film, since glass only minimally absorbs electromagnetic radiation in the near infrared range below 3 μm wavelength. This glass plate is fixed by a frame or tensioning collar 34 and oriented parallel to the radiation source 31. The distance is also determined by the focus position of the laser on the polymer body 37.

[0050] By means of an axis system with the axes 32 and 33, the radiation source 31 can be moved parallel to the polymer main body 37 and thus traverse the contours to be welded. The power and rate of advance of the laser can be variably adjusted.

[0051] The movement of the cylinder 39 relative to the radiation source 31 is realised via at least two axes 32 and 33, which move either the cylinder 39 via a traveling table 45 or the radiation source 31 in space.

[0052] The flexible diaphragm 44 can be tensioned in parallel over the microfluidic main body 37 by means of a bracing apparatus.

[0053] The flexible diaphragm can be tensioned in various ways. In order to achieve a plane-parallel application of the film on the glass plate to the best possible extent, it is possible to etch micro channels 46 into the glass plate 35 (FIG. 10 with the vacuum chamber 47 made of glass) via selective laser etching (Meineke et al. 2016), thus making it possible to create a vacuum in these channels by a connected vacuum pump and thus to suck the flexible diaphragm against the glass plate (FIG. 11) before the microfluidic main body is pressed against it. This reduces unevennesses of the flexible diaphragm.

[0054] Another tensioning option uses magnets which are embedded in the piston table. The film is pre-tensioned by hand over the main body and then held in place by further oppositely poled magnets. The magnets are mounted on a fixable rail that can be moved in one direction so that the diaphragm film can be stretched further and then fixed in the desired position (FIG. 4). This increases the tensioning precision.

[0055] Other ways of tensioning the flexible diaphragm are pneumatic cylinders. Here, the diaphragm is fixed on one side (e.g. with magnets), then tensioned over the main body and fixed on the opposite side with a pneumatic cylinder, this cylinder being fixed on an orthogonally mounted further cylinder, so that the diaphragm can be further stretched or tensioned by extending the cylinder in the x direction with defined force development. This results in a homogeneous tension over the entire welding area.

[0056] The polymer main body contains microstructures which, in their entirety, form a plurality of pumps and valve systems in interaction with the diaphragm film. A large number of valves, pump chambers and channels, as well as inlets and outlets, create a microfluidic array which enables the transport of liquid or gas from fluid inlets individually to the microreactors.

[0057] Such an array can consist of an actuator terminal block, as described in the European patent EP3055065, and the microreactor array with integrated microfluidic chip. The microfluidic chip consists of valves consisting of a spherical segment with a concentric line seal and a flexible diaphragm. Microchannels lead to the centre of the valve and to the circumference of the spherical segment. The flexible diaphragm can be moved via an actuator and can be closed and opened.

[0058] The control of the individual diaphragm valves can be realised by different methods. Among others, pneumatic control channels are possible here, but optically, thermally, hydraulically, electromechanically or magnetically activated switches can also be used for fluid channel control.

[0059] One possibility is to create a peristaltic movement, in which the fluid is first pressed through the inlet into the open inlet valves and the open pump chamber. By subsequently closing the inlet valves, a precise volume of fluid is trapped within the pump chamber. By opening an outlet valve and closing the pump chamber, the volume of the pump chamber can be conveyed in the direction of the channel outlet (FIGS. 1 to 3. The volume conveyed is largely determined by the precision of the pump chamber, which is generated from the structure of the polymer body and the covering by the diaphragm film. With this technique it is also possible to control a plurality of fluidic channels via one inlet and one pump chamber (FIG. 11).

[0060] The described invention significantly increases the precision of the valve covering; primarily, it reduces the variation in the volume of the volume enclosed by the valve trough and cover film, thus improving the precision of the dosing process. The mechanistic reason for this is that laser transmission welding allows a more precise geometry of the weld edge—or weld seam. This is achieved by a strictly locally limited energy input and thus softening of the substrate only at precisely defined points or along precisely defined seams. An unintentional relevant heat transfer outside the defined areas, especially an energy input into the pump/valve trough is thus almost completely avoided.

[0061] The polymer body (m2p-labs GmbH, Baesweiler, MTP-MF32-BOH 1 from Topas®) is fixed on the positioning table as described and the diaphragm film (Topas® ELASTOMER E-140, 100 μm thickness) is tensioned over the area to be welded. An example of the valve contour before welding is shown in FIG. 12. Using a CAD program (e.g. Autodesk AutoCAD), a corresponding weld contour is created (e.g. FIG. 13 weld contour). This weld contour can then be loaded into the welding program. The speed of travel at individual points, the beam power and the position for activating and deactivating the laser can also be set. The body to be welded is then pressed against the glass plate 35 via the cylinder (Festo ADN-100-60-A-P-A) at a pressure of 0.1 to 5 bar, preferably 0.75 bar. Too high a pressure causes the diaphragm film to deform and be pressed into the valves. Too low a pressure slows down the flow of material within the weld seam and thus reduces the strength of the weld seam. By reading out the force sensors (ME-Messtechnik KM26), it is ensured that the force distribution is homogeneous or must be readjusted via the adjusting screws. An inhomogeneous force distribution leads to an inhomogeneous focusing of the laser.

[0062] For welding, a thulium fibre laser of the company “IPG Laser” with the wavelength 1940 nm can be used. This wavelength is suitable because the polymer used (COC, cycloolefin copolymer; a copolymer of norbornene and ethene) is absorbent in this wavelength range. A corresponding optical system with a focal length of 20 mm focuses the laser beam. Depending on the rate of advance, a laser power of 2 to 50 W is required for the welding process; 5 to 25 watts at a rate of advance of the laser of 10 mm/min to 2000 mm/min are preferred, and 8 watts at a feed rate of 200 mm/min are particularly preferred. The required moderate laser power allows the choice between a large number of lasers, such as a thulium fibre laser from Keopsys (CW_Laser CTFL-TERA) or the IPG laser (TLM-200 Thulium CW Fiber Laser Module).

[0063] However, the method described here can be used not only for COC (Topas®) but also for other polymers that absorb in the infrared range. Examples are polystyrene, polymethyl methacrylate, polycarbonate, polyethylene etc.

[0064] The radiation source can be moved over the welding area in x, y and z direction via an axis system (e.g. Bosch Rexroth linear systems) in order to move the individual valve, channel and pump contours at a speed of, for example, 200 mm/min at a laser power of approximately 8 W. The laser beam is only activated here at the designated contours, thus avoiding unwanted energy input. With a focal length of 20 mm, the radiation source is positioned at a height of approximately 17 mm relative to the polymer body surface. This height must be adjusted by changing the focal length. In order not to melt the channels by increased local energy input, the weld contour must be generated at a precise distance from the channel of approximately 0.3 mm FIG. 14 shows a valve contour with weld seam.

[0065] The flexible diaphragm is softened only at the points where the laser beam penetrates and is connected to the main body by thermal fusion. Due to the high travel speed of the radiation source, melting of the valve or channel contours is prevented and the weld seam is defined. A variation of the beam power can further influence this seam. A high precision of the valve contour can thus be achieved by a sufficient accuracy of the axis system. This is directly reflected in the precision of the dosing process.

[0066] To measure the precision of the flow, the microfluidic chip is adhesively bonded as the bottom of a 48-well microtiter plate in an air- and liquid-sealed manner. The microtiter plate is placed on an orbital shaker, which mixes the liquids inside the microtiter plate at up to 1500 rpm (revolutions per minute). The transparency of the polymer bottom or microfluidic chip allows optical measurements of the liquid inside each individual reaction chamber. For example, fluorescence signals of green fluorescent protein, fluorescein or riboflavin can be detected. Such a measurement setup is implemented in the BioLector Pro from m2p-labs GmbH, Baesweiler, Germany.

[0067] To measure the flow rate, a mixture of 50 mM aqueous buffer solution (K2HPO4) was filled with 70 μM fluorescein via the channel input of the microfluidic chip described in EP3055065. Using an optical waveguide and corresponding optical filters with an excitation wavelength of 436 nm and a detection wavelength of 540 nm and the evaluation electronics of the BioLector Pro from m2p-labs, even the smallest changes in fluorescence in the reaction chambers above the microfluidic plate can be detected. The fluorescein-containing buffer solution is conveyed from the channel inlet in the reservoir well to the channel outlet in the reaction chamber via the described actuator of the pumping process. In the reaction chamber, 800 μL of a buffer solution consisting of 50 mM K2HPO4 is supplied. The BioLector Pro has 16 reservoir wells and 32 reaction chambers. From each reservoir well, solution can be conveyed through the bottom of the microfluidic plate into four reaction chambers. If the same fluorescein-containing buffer solution is filled into all reservoir wells and all pumps and valves in the microfluidic chip are controlled in the same way, the fluorescein solution is conveyed in the same way into all 32 reaction chambers. This arrangement thus makes it possible to check whether all pumps and valves that deliver the fluorescein solution from the reservoir wells to the reaction vessels convey the fluorescein solution in the same way and uniformly. This can be quantified by measuring the intensity of the fluorescence of the fluorescein pumped from the reservoir wells into the reaction chambers at regular intervals in all reaction chambers and by determining the change in fluorescence over time. This measurement can also be performed fully automatically in the BioLector Pro. With 0.5 bar pneumatic pressure, the liquid is pumped into the microfluidic channel via the inlet valves and the pump chamber to the outlet valves. The inlet valves close at 2 bar. Opening the outlet valves allows the fluorescein solution to enter the corresponding associated reaction chamber. By closing the pump chamber at 2.5 bar, the liquid is conveyed into the corresponding reaction chamber. The outlet valve is then also closed pneumatically at a pressure of 1.5 bar. This pumping process is repeated continuously for all 32 reaction chambers, resulting in a flow of 5 μL/h per reaction chamber.

[0068] Over a period of about 20 h, the change in the fluorescence signal of all 32 reaction chambers of the microtiter plate is recorded. After completion of the measurement, the mean value of the change in the fluorescence signal of all 32 measured values and the corresponding standard deviation as well as the relative standard deviation are determined. If all microfluidic pumps in the chip have been controlled in the same way, an identical flow rate into all 32 reaction chambers would be expected, and accordingly a standard deviation of zero. Higher standard deviations are an indicator for differences between the pumps or their control, which result in variations of the flow rate.

[0069] The test was carried out several times both with microfluidic chips where the diaphragm film is applied by thermofusion, and with microfluidic chips where the diaphragm film has been connected to the polymer main body by laser welding. The results show for the microfluidic chips manufactured by laser welding, compared to the microfluidic chips manufactured by thermofusion bonding, a significant increase of the precision of the pumping process, or rather a significant reduction of the standard deviation of the gradient of the fluorescence signal. The relative standard deviation of the change in the fluorescence signal over time was 12% on average in the case of chips produced by thermofusion bonding; for laser-welded chips it was less than 7% on average.

[0070] The above-mentioned components as well as components claimed and described in the embodiments to be used in accordance with the invention are not subject to any special exceptional conditions with regard to their size, shape, design, material choice and technical concepts, and therefore the selection criteria known in the field of application can be applied without restriction.