Abstract
A method for diluting, mixing, and/or aliquoting two liquids using a microfluidic system with at least two pump chambers first includes filling at least one of the at least two pump chambers with a first liquid. The at least two pump chambers are connected to one another by at least one microfluidic channel. The method then includes pumping the first liquid through the channel. The at least one channel is configured such that at least part of the first liquid remains in the channel after the pumping. The method then includes determining the part of the first liquid that remains in the channel. The method then includes flushing the channel with a second liquid.
Claims
1. A method of diluting, mixing and/or aliquoting two fluids using a microfluidic system with at least two pump chambers connected to one another by at least one microfluidic channel, the at least one channel configured such that at least a portion of the first fluid remains in the channel after a pumping operation, the method comprising: filling at least one of the at least two pump chambers with a first fluid; pumping the first fluid through the channel, leaving a portion of the first fluid in the channel; ascertaining the portion of the first fluid remaining in the channel; and purging the channel with a second fluid.
2. The method as claimed in claim 1, wherein the portion of the first fluid remaining in the channel is ascertained in order to monitor the attainment of one or more of a desired mixing ratio and a desired dilution level.
3. The method as claimed in claim 1, wherein the portion of the first fluid remaining in the channel is ascertained in order to ascertain a volume of the second fluid required for the desired dilution, mixing, and/or aliquoting.
4. The method as claimed in claim 1, wherein the portion of the first fluid remaining in the channel is ascertained from a volume of the channel.
5. The method as claimed in claim 1, wherein the portion of the first fluid remaining in the channel is ascertained by signals from a camera surveying the channel.
6. The method as claimed in claim 1, wherein the channel is filled completely with the first fluid after the pumping operation.
7. The method as claimed in claim 1, wherein the at least two chambers are emptied before the channel is purged with the second fluid, and wherein the first fluid remains in the channel after the at least two chambers have been emptied.
8. A computer program configured to execute a method of diluting, mixing, and/or aliquoting two fluids using a microfluidic system with at least two pump chambers connected to one another by at least one microfluidic channel, the at least one channel configured such that at least a portion of the first fluid remains in the channel after a pumping operation, the method includes: filling at least one of the at least two pump chambers with a first fluid, pumping the first fluid through the channel, leaving a portion of the first fluid in the channel, ascertaining the portion of the first fluid remaining in the channel, and purging the channel with a second fluid.
9. The computer program as claimed in claim 8, wherein the computer program is stored on a machine-readable storage medium.
10. An electronic control device configured to dilute, mix, and/or aliquot two fluids using a microfluidic system with at least two pump chambers connected to one another by at least one microfluidic channel, the at least one channel configured such that at least a portion of the first fluid remains in the channel after a pumping operation, the electronic control device configured to operate the microfluidic system to: fill at least one of the at least two pump chambers with a first fluid, pump the first fluid through the channel, leaving a portion of the first fluid in the channel, ascertain the portion of the first fluid remaining in the channel, and purge the channel with a second fluid.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Working examples of the invention are illustrated in the drawings and is elucidated in detail in the description that follows.
[0016] FIGS. 1 a-d show schematic diagrams of a first embodiment of the invention.
[0017] FIGS. 2 a-f show schematic diagrams of a second embodiment of the invention.
[0018] FIGS. 3 a-i show schematic diagrams of a third embodiment of the invention.
[0019] FIGS. 4 a-g show schematic diagrams of a fourth embodiment of the invention.
[0020] FIG. 5 shows a schematic diagram of a lab-on-chip on which one embodiment of the method of the invention can proceed.
WORKING EXAMPLES OF THE INVENTION
[0021] FIGS. 1 a-d show schematic diagrams of a microfluidic system. Elucidated hereinafter is the basic concept of the microfluidic system that can be regarded as an independent module for use in a microfluidic network. The parts of the FIG. 1 a-c each show a step of a first embodiment of the method of the invention. The microfluidic system has a first pump chamber 1 and a second pump chamber 2, which, in this working example, are each of the same construction and each have a membrane (not shown) which is deflected in the course of pumping and displaces a fluid from the respective chamber. In further working examples, the first pump chamber 1 and the second pump chamber may differ in construction, function, volume and the components encompassed. The two chambers 1, 2 are connected to one another via a microfluidic channel 3, wherein the microfluidic channel 3 connects an outlet 11 of the first chamber 1 to an inlet 20 of the second chamber 2. The volume of the pump chambers 1, 2 is, for example, 30 μl, and the ratio between the volume of the pump chambers 1, 2 and the volume of the channel is 1:100. In FIG. 1a, the first chamber 1 has been filled via its inlet 10 with a first fluid F.sub.1 which includes an analyte to be analyzed and is based on water. The second chamber 2 and the channel 3 have been filled with a second fluid F.sub.2.
[0022] In FIG. 1b, the membrane of the first chamber 1 is deflected and hence the first fluid F.sub.1 is pumped through the channel 3 into the second chamber 2, and this displaces the second fluid F.sub.2 from the second chamber 2 and the channel 3. The channel 3 is configured such that at least a portion of the first fluid F.sub.1 remains in the channel 3 after the pumping operation. It is surface effects in particular, for example the surface tension of the first fluid F.sub.1 itself and the interfacial tension between the first fluid F.sub.1 and the surface of the channel 3 in contact with the first fluid F.sub.1, that act here on the first fluid F.sub.1 and lead to a capillary effect of the first fluid F.sub.1 in the channel 3, as a result of which this is retained in the channel 3. The surface effects mentioned are dependent on the geometry, shape and length of the channel 3, the material of the surface of the channel 3, and the fluid F.sub.1 itself. In this working example, the channel 3 is filled completely with the first fluid F.sub.1 after the pumping operation.
[0023] In FIG. 1c, the second chamber 2 is emptied via its outlet 21, with the first fluid F.sub.1 still remaining in the channel 3 even after the emptying owing to the configuration of the channel 3 and the active surface effects. Subsequently, a second fluid F.sub.2 with which the first fluid F.sub.1 is to be mixed or diluted is introduced into the microfluidic system via the inlet 10 of the first chamber 1 and flushed through the channel 3. In FIG. 1d, the first fluid F.sub.1 and the second fluid F.sub.2 mix to give a first mixture M.sub.1 and the first fluid F.sub.1 is diluted with the second fluid F.sub.2. Since the geometry, shape and length of the channel 3 is known and this was filled completely with the first fluid F.sub.1, it is possible to use these to ascertain the volume of the first fluid, such that the mixing ratio or dilution level can be controlled. Moreover, a the aliquoting, i.e. ascertaining of the proportion of the first fluid F.sub.1 or of the analyte, is envisaged.
[0024] In FIGS. 1-4, for reasons of clarity, the representation of valves for control of the fluid flow is dispensed with. Identical components are referred to by the same reference numerals hereinafter, and so repeated description thereof is dispensed with. The designations “first chamber” and “second chamber” are based on their filling with a first fluid F.sub.1 and a second fluid F.sub.2, respectively. Within the parts of the figures, for better clarity, reference numerals for fixed components are included only in the respective parts a of the figures and can be applied to the further parts of the figures.
[0025] FIGS. 2 a-f show schematic diagrams of a microfluidic system in which the outlet 11 of the first chamber 1 and the outlet 21 of the second chamber 2 are connected via a microfluidic channel 3, and the inlet 10 of the first chamber 1 and the inlet 20 of the second chamber 2 are connected via a further microfluidic channel 3′ of analogous design to the microfluidic channel 3, such that the microfluidic system forms a closed microfluidic circuit. The channel 3 is connected to a common outlet 30. By means of the inlets 10, 20 and the common outlet 30, the above-described module can be incorporated into a microfluidic network. The chambers 1, 2, inlets 10, 20, and the common outlet 30 may be actuated individually.
[0026] The parts of the FIGS. 2 a-f each show a step of a second embodiment of the method of the invention. In FIG. 2a, the first chamber 1 has been filled with a first fluid F.sub.1, and a second fluid F.sub.2 is being held in the second chamber 2. In FIG. 2b, the first fluid F.sub.1 is pumped out of the first chamber 1 into the channel 3′ and through the outlet 30. As described above, a portion of the first fluid F.sub.1 remains in the channel 3. In FIG. 2c, the two chambers 1, 2 are alternately opened and closed while pumping, such that the second fluid F.sub.2 moves through the channels 3, 3′ in a closed circuit and mixes with the first fluid F.sub.1 that has remained in the channel 3. This operation is referred to as cyclical mixing. After a defined number of cycles, the two fluids F.sub.1 and F.sub.2, as shown in FIG. 2d, are mixed completely to give a mixture M.sub.1 with a defined mixing ratio, and the chambers 1, 2 are closed. The mixture M.sub.1 can then be used for further analysis purposes. FIGS. 2e and 2f show how a further mixture M.sub.2 with a different mixing ratio is produced from the mixture M.sub.1. Similarly to the first fluid F.sub.1, a portion of the first mixture M.sub.1 also remains in the channels 3, 3′. The second chamber 2 is filled again with the second fluid F.sub.2 via the inlet 20. In other working examples, depending on the desired mixing ratio, the first chamber 1 may instead be filled with the second fluid F.sub.2, or either the first chamber 1 or the second chamber 2 may be filled with the first fluid F.sub.1. This is in turn followed by the cyclical mixing of the second fluid F.sub.2 with the first mixture M.sub.1 which have been mixed completely to give the second mixture M.sub.2 after a defined number of cycles in FIG. 2f.
[0027] FIGS. 3 a-i show schematic diagrams of a microfluidic network with which dilution series with different dilution levels and mixing ratios can be achieved. The outlet from the first chamber 1 is connected via the microfluidic channel 3 simultaneously to the inlet 20 of the second chamber 2 and an inlet 40 of a third chamber 4. The outlet 21 of the second chamber 2 is connected via a further microfluidic channel 3′ of analogous design to the microfluidic channel 3 to an outlet 41 of the third chamber 4. The further channel 3′ has a common outlet 30 and branches repeatedly and hence forms a network. Each branch of the common outlet 30 and the chambers 1, 2, 4 may be actuated individually by means of the valves described at the outset. At the point in the common outlet 30 identified by reference numeral 31 is disposed a bypass (not shown in detail). This bypass can likewise be used to purge at least the common outlet 30.
[0028] The parts of the FIGS. 3 a-i each represent a step of a third embodiment of the method of the invention. In FIG. 3a, the first chamber 1 has been filled with the first fluid F.sub.1 which, in FIG. 1b, is pumped through the channel 3 into the third chamber 4, leaving a portion of the first fluid F.sub.1 in the channel. The chamber 1 is filled with the second fluid F.sub.2 and the latter is subsequently, as shown in FIG. 3c, pumped into the second chamber 2, here too leaving a portion of the second fluid F.sub.2 in the channel. In FIG. 3d, the second fluid F.sub.2 is cyclically mixed with the first fluid F.sub.1, as already elucidated in connection with FIG. 2c, which affords a first mixture M.sub.1 having a defined mixing ratio and a defined dilution level. The first mixture M.sub.1 is, as shown in FIG. 3e, diverted into one of the branches by the outlet 30 and can then be used further. Subsequently, the common outlet 30 is purged via the abovementioned bypass, such that the first mixture M.sub.1, apart from a negligibly small portion, is removed from the common outlet 30. FIG. 3f combines multiple steps. A portion of the first mixture M.sub.1 remains here in the channel 3′, and this is then pumped into the second chamber 2. Moreover, the first chamber 1, in analogy to FIG. 3a, is filled again with the first fluid F.sub.1, and the first fluid F.sub.1, in analogy to FIG. 3b, is then pumped again into the third chamber 4. Depending on the desired mixing ratio and the dilution level, in further working examples, the second fluid F.sub.2 can be used instead. In FIG. 3g, the first mixture M.sub.1 is again cyclically mixed with the first fluid F.sub.1 in order to obtain a second mixture M.sub.2. This second mixture M.sub.2 is then, as shown in FIG. 3h, diverted into a further branch by the outlet 30. The aforementioned steps are repeated in order to obtain the dilution series, shown in FIG. 3i, with eight different mixtures M.sub.1, . . . M.sub.8 that each have a different mixing ratio and different dilution levels.
[0029] FIGS. 4 a-g show schematic diagrams of a microfluidic system for performance of a nested PCR (polymerase chain reaction). The intention here is to divide a pre-amplicon into two different reaction strands and to dilute primers of the pre-amplicon to such a degree that they are no longer active in a second PCR. The first chamber 1 and second chamber 2 already described are each assigned a further chamber 5, 6 in which there are lyophilizates L, also called lyobeads. The chambers 1, 2, 5, 6 are connected to one another via microfluidic channels 3. The first chamber 1 and the second chamber 2 together form a circuit. In addition, the first chamber 1 with its accompanying chamber 5 and the second chamber 2 with its accompanying chamber 6 each form a sub-circuit. In order to be able to achieve what is called shuttle PCR, the first chamber 1 and the second chamber 2 may be assigned further chambers that are not shown in further working examples, such that three chambers in each case form a unit.
[0030] The parts of the FIGS. 4 a-g each show a step of a fourth embodiment of the method of the invention. At the start, in FIG. 4a, the first chamber 1 is filled, for example, with the reaction product of a pre-amplification as first fluid F.sub.1. The first fluid F.sub.1 is then, in FIG. 4b, pumped through the circuit between the first chamber 1 and the second chamber 2. A portion of the first fluid F.sub.1 remains in the channel 3. Subsequently, the microfluidic system is purged with an aqueous second fluid F.sub.2, as shown in FIG. 4c. The first fluid F.sub.1, i.e. the pre-amplicon, and the second fluid F.sub.2, i.e. the buffer, are then, as shown in FIG. 4d, mixed by pumping the first chamber 1 and the second chamber 2 in circulation, giving rise to a mixture M.sub.1. The dilution level can be adjusted by repeating the steps as described in connection with FIG. 3. If the desired dilution level has been attained, the mixture M.sub.1 is pumped into the chambers 5 and 6—see FIG. 4e. By pumping the mixture M.sub.1 in the sub-circuit between the first chamber 1 and the associated chamber 5, and in the sub-circuit between the second chamber 2 and the associated chamber 6, the lyophilizates L present therein are dissolved in the mixture M.sub.1—see FIG. 4f. The mixed product obtained is then, as shown in FIG. 4g, pumped into the first chamber 1 and into the second chamber. Subsequently, the second, specific PCR is started.
[0031] FIG. 5 shows a schematic diagram of a lab-on-chip with a feedback system on which one embodiment of the method of the invention can proceed. The microfluidic system S is surveyed by a camera 7 that records the fluorescence and/or turbidity of the fluid. Additionally provided is an evaluation unit 8 that receives the camera signals. The evaluation unit 8 uses an algorithm to ascertain the dilution level and/or the mixing ratio of the fluids F.sub.1, F.sub.2 from the camera signals. The above-described steps for altering the dilution level are repeated until the desired dilution or the desired mixing ratio has been attained and recognized by the evaluation unit 8. Then the evaluation unit 7 transmits an approval signal to a pneumatic control unit 9 that controls the microfluidic system S and triggers further steps, for example the onward conduction of the mixture obtained. In addition, the evaluation unit 8 ascertains the volume of the second fluid F.sub.2 required for the desired dilution, mixing and/or aliquoting. Firstly, the evaluation unit 8 can compare the dilution level with pre-calibrated dilution levels. Secondly, the evaluation unit 8 can be calibrated during the analysis. The volume of the portion of the first fluid F.sub.1 remaining in the channel 3 is ascertained from the geometry, shape and length of the channel 3, and the volume of the second fluid F.sub.2 is either measured externally or likewise ascertained via the portion remaining in the channel 3. The evaluation unit 8 uses the two volumes of the two fluids F.sub.1 and F.sub.2 to calculate the dilution level or the mixing ratio, and associates them with the camera signals. Thirdly, a reference fluid with known dilution level or mixing ratio can be introduced into the second chamber 2. Then the evaluation unit 8 compares the mixture of the two fluids F.sub.1 and F.sub.2 with the reference fluid. In addition, the evaluation unit 8 can record the dilution levels or mixing ratios, which can then be incorporated into an analysis algorithm of a corresponding assay.
