Centrifuge Unit for a Microfluidic Device for Processing Liquid Samples

Abstract

A centrifuge unit for a microfluidic device for processing liquid samples includes a centrifuge cup and at least one centripetal force-dependent valve function.

Claims

1. A centrifuge unit for a microfluidic device for the processing of liquid samples, the centrifuge unit comprising: a centrifuge cup; and at least one centripetal force-dependent valve arrangement.

2. The centrifuge unit as claimed in claim 1, wherein the centripetal force-dependent valve arrangement is a reversible valve arrangement.

3. The centrifuge unit as claimed in claim 1, wherein the centripetal force-dependent valve arrangement includes at least one sealing ring.

4. The centrifuge unit as claimed in claim 3, wherein the sealing ring externally surrounds the centrifuge cup and reversibly exposes or closes openings in the centrifuge cup as a function of an acting centripetal force.

5. The centrifuge unit as claimed in claim 1, wherein the valve arrangement comprises: at least one opening defined in the wall of the centrifuge cup; a bolt movably arranged in the at least one opening and passing through the wall, the bolt having a bolt head arranged outside the centrifuge cup and a sealing ring below the bolt head, wherein, below a predeterminable centripetal force, the bolt is held within the centrifuge cup is held in a sealing position by a secured compression spring.

6. The centrifuge unit as claimed in claim 5, wherein the compression spring is secured by a locknut which is mounted in an interior of the centrifuge cup on a thread of the bolt.

7. The centrifuge unit as claimed in claim 1, wherein the valve arrangement is an irreversible valve arrangement function.

8. The centrifuge unit as claimed in claim 7, wherein the irreversible valve arrangement is actuated at a higher centripetal force than an additional reversible valve arrangement.

9. The centrifuge unit as claimed in claim 1, wherein the centrifuge cup is equipped at least partly with surface structures configured for adherence of sample constituents.

10. A microfluidic device for the processing of liquid samples, comprising: at least one centrifuge unit comprising: a centrifuge cup; and at least one centripetal force-dependent valve arrangement.

11. The device as claimed in claim 10, wherein the centrifuge cup is configured to be arranged at a tilt angle between 10 and 50° during the processing.

12. The device as claimed in claim 10, further comprising: an integration position within the microfluidic device configured to receive the centrifuge unit, the integration position having a further fluidic entry.

13. A method for operating a centrifuge unit having a centrifuge cup and at least one centripetal force-dependent valve arrangement in a microfluidic device, the method comprising: after introduction of the sample into the centrifuge cup, rotating the centrifuge cup at a first speed in a first phase and rotating the centrifuge cup at a second, increased speed in a subsequent second phase, wherein the increased speed causes actuation of the at least one centripetal force-dependent valve arrangement of the centrifuge unit.

14. The method as claimed in claim 13, further comprising: refilling the centrifuge cup repeatedly by flooding the centrifuge gup with a liquid.

15. The method as claimed in claim 13, further comprising: removing cellular constituents of a sample and/or lyzing cellular constituents and/or selectively isolating particular cellular constituents of a sample via the rotation of the centrifuge cup at the first speed and the rotation of the centrifuge cup at the second speed.

Description

[0025] In the Drawings:

[0026] FIG. 1 shows a schematic section through a part of a microfluidic device with integrated centrifuge unit according to a first embodiment;

[0027] FIG. 2 shows a schematic section of the embodiment from FIG. 1 to illustrate a rinsing procedure;

[0028] FIG. 3 shows a schematic section of a further embodiment of a centrifuge unit;

[0029] FIG. 4 shows a schematic plan view of a centrifuge unit in a further preferred embodiment; and

[0030] FIG. 5 shows a schematic section of a further preferred embodiment of a centrifuge unit with reversible and irreversible valve functions.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0031] FIG. 1 shows, schematically, the integration of the centrifuge unit with the centrifuge cup 10 within a sample input chamber 101 of a microfluidic device 100. The centrifuge cup 10 is mounted rotatably between a lower sealing layer 102 and a structuring fluidic layer 103 of the device, by means of a spindle 11, which may preferably be metallic or else may be made, for example, of plastic. The sealing layer 102 and the fluidic layer 103 are made of polycarbonate, for example. The spindle 11 is inserted into corresponding indentations or abutments of the layers 102 and 103. In this embodiment the centrifuge cup 10 is closed off internally in the upper region by a sealing ring (O-ring) 12. This sealing ring 12 realizes a centripetal force-dependent valve function. The liquid 1 within the centrifuge cup 10 is rotated together with the centrifuge cup 10. The liquid 1 may be, for example, a blood sample, introduced directly into the centrifuge cup 10 beforehand via the sample inlet opening 104 in arrow direction. The actual drive for the rotation of the centrifuge cup 10 may be accomplished in a variety of ways, as for example by a direct mechanical intervention of a drive motor by mechanical “drivers” or by a magnetic coupling or a magnetic rotational field, or pneumatically.

[0032] During operation, the centrifuge cup 10 is arranged preferably with a particular tilt angle, preferably with a tilt angle of about 30°. This tilt angle may be realized, for example, by positioning of the entire microfluidic device 100 at a corresponding angle in a processing apparatus. The rotation of the centrifuge cup 10 produces centripetal forces, and so the sample liquid 1 lies against the wall of the centrifuge cup 10 with a certain pressure. Up to a certain threshold value, the sealing face between the sealing ring 12 and the wall of the centrifuge cup 10 withstands the prevailing pressure, allowing centrifugation to take place over a certain time at the corresponding speed until, for example, all of the cellular constituents have deposited on the wall of the centrifuge cup 10. When this state has been reached, the speed of the centrifuge cup 10 is increased, and so the threshold pressure is exceeded at the sealing face and the liquid freed from cellular constituents (for example, the plasma after centrifugation of a whole blood sample) is pressed through the sealing face between sealing ring 12 and internal wall of the centrifuge cup 10. The centrifugate—the plasma, for example—is spun into the sample input chamber 101 and can then be coupled via a sample chamber outlet 105 into the fluidic network of the microfluidic device 100. Provision may optionally be made for the liquid to be collected before the exit 105.

[0033] Opening into the sample input chamber 101, below the centrifuge cup 10, is a further fluidic entry 106, with which, for example, the region in which the centrifugate spins out and/or the sample chamber outlet 105 and/or the centrifuge cup 10 itself are cleaned and/or rinsed with appropriate medium, as illustrated in FIG. 2. Via the fluidic entry 106 a further liquid 2 is introduced from below into the sample input chamber 101. The liquid level of the liquid 2 here may rise above the lower-lying edge of the centrifuge cup 10 in the sample input chamber 101, and so the liquid 2 penetrates into the centrifuge cup 10 and refills it. The degree of filling of the centrifuge cup 10 can be controlled by the amount of the liquid 2 supplied. The 30° degree inclination of the centrifuge cup 10 is particularly useful here, since this inclination causes the liquid 2 to cross into the centrifuge cup 10 at a defined location, thereby making it possible, for example, to prevent the centrifuge cup 10 being filled with excessive liquid. For a rinsing process, the liquid 2 can be centrifuged out of the centrifuge cup 10 again subsequently. The cycle of filling and centrifuging (rinsing) may be repeated multiply one after another, and, depending on particular application, the liquids which have been centrifuged off may each be wholly or partly processed further in the microfluidic device or else discarded.

[0034] FIG. 3 shows a further, particularly preferred embodiment of a centripetal force-dependent valve function. In this case an externally peripheral sealing ring 22 is provided in the upper region of the centrifuge cup 20, and closes openings in the wall of the centrifuge cup 20. The centrifuge cup 20 is equipped toward the top with an internally protruding rim 23, in order to prevent the liquid 1 escaping from the centrifuge cup 20 at a low speed. This embodiment with external sealing ring 22 has the advantage that the centripetal forces Fc acting on the sealing ring 22 stretch the ring away from the sealing face and/or reduce its sealing force with respect to the external face of the centrifuge cup 20. Accordingly a greater proportion of the liquid 1 can be centrifuged off from the centrifuge cup 20, since, in comparison with the embodiment in FIGS. 1 and 2, the mass of liquid is no longer the only determining factor for the pressure conditions at the seal. With particular preference the sealing ring 22 and the rotational forces are harmonized with one another in such a way that the centripetal forces alone are sufficient to open the valve formed by sealing ring 22 and external wall of the centrifuge cup 20. This has the particular advantage that the entire quantity of liquid can be centrifuged off without residues remaining in the centrifuge cup 20. For this purpose a relatively supple material (e.g., a supple rubber ring) is usefully used for the sealing ring 22, readily stretchable by exposure to the centripetal force. The embodiment with an external sealing ring 22, relative to an internal sealing ring in accordance with the embodiment in FIGS. 1 and 2, has the advantage that, even when speeds are high and there is little liquid in the centrifuge cup 20, it is still possible for liquid to pass through the sealing face between sealing ring 22 and the wall of the centrifuge cup 20.

[0035] FIG. 4 shows a particularly preferred exemplary embodiment of a centripetal force-dependent valve function 35 for a centrifuge unit. Shown here in plan view are three valve facilities 35, integrated into the wall of the centrifuge cup 30. The valves 35 distributed over the periphery of the centrifuge cup 30 are formed by a bolt 36 which passes through the wall of the centrifuge cup 30. The openings provided for this purpose in the centrifuge cup 30 are somewhat larger than the diameter of the respective bolt 36, so that the bolt 36 is freely movable within the opening. On the inside of the centrifuge cup 30, the bolt 36 is provided with a compression spring (return spring) 37 and a retaining nut 38 or other securement. Located on the external side of the valve 35 is a bolt head 39 on the bolt 36. Between bolt head 39 and the wall of the centrifuge cup 30 there is a peripheral sealing ring 32, which seals off the opening externally. By means of the compression spring 37, which presses in the direction of the retaining nut 38, the bolt head 39 is tightened on the outside sealing ring and presses it against the external wall of the centrifuge cup 30. This results in tight closure of the passage opening. When the centrifuge cup 30 is rotated, the centripetal force Fc acts against the spring force of the compression spring 37. At a particular speed, the centripetal force Fc is sufficient to expose the openings for the passage of liquid, and so the liquid centrifuged in the centrifuge cup 30 emerges from the centrifuge cup 30 and is able to pass into the microfluidic network of the device, which is not shown in more detail here.

[0036] The speed threshold which must be achieved for actuation of the valve function 35 in this way is determined by the mass of each bolt 36 with the locknut 38 located on it, and also by the spring force of the compression spring 37 which counteracts it. A part is additionally played by the position of the locknut 38 in relation to the bolt 36. If the bolt 36 is provided with a thread, the locknut 38 can easily be altered in its position, thereby enabling adjustment of the pretension of the compression spring 37 and hence its retaining force. Biocompatible materials in particular are suitable materials for the individual components of the valve construction 35. For example, stainless steel socket-head screws M4×12 with M4 nuts can be used, together with a silicone O-ring (e.g., 4×2 mm) and steel springs. These illustrative dimensions are suitable for centrifuge units in which the centrifuge cup has a diameter, for example, of 3-4 cm. For smaller centrifuge units, which are suitable especially for commercially customary microfluidic devices, and which preferably have a centrifuge cup diameter of about 5 to 20 mm, more particularly 5 to 15 mm, 10 mm for example, the dimensions chosen for the components for the valve function should be reduced accordingly. Alternatively to screws it is possible to use threadless bolts, and alternatively to nuts it is possible to use, for example, tap rings or circlips or self-retaining clamping rings, which are placed on and/or pressed on.

[0037] Through the choice of suitable components and materials, for example, the valve function may be configured such that the valves open and the transfer of liquid can take place at a speed of 10 000 rpm. The diameter chosen for the openings within the wall of the centrifuge cup may be 1 mm, for example. It is possible, for example, to use three openings each of 1 mm in the periphery of the centrifuge cup. For different applications it may be advantageous to keep the openings for the passage of liquid relatively small, possibly even smaller than 1 mm in diameter, in order to prevent the transfer of cells. This may be sensible in particular when a centrifuge unit is to be used to obtain cell-free plasma from whole blood. Another result of relatively small openings may be that there is no unwanted centrifugal removal of cells, in those applications, for example, in which erythrocytes in whole blood are first selectively lyzed, centrifuged off and discarded, before other blood cells, leucocytes, for example, which have been retained in the centrifuge cup are to be lyzed and, for example, their nucleic acids extracted and processed further.

[0038] Relatively small openings have the disadvantage, however, that even at high speeds the complete transfer of the liquid from the centrifuge cup to the outside may require a certain time interval, 1-2 minutes for example. This may be a drawback in those cases in which during this time interval a previously resuspended cell material would be centrifuged again against the internal walls of the centrifuge cup. For such cases in particular, though also generally for cases in which rapid transfer of liquid is to take place for other reasons, relatively large openings may be advantageous for the transfer of liquid. Provision may be made, for example, for relatively large openings in the centrifuge cup to be provided additionally, in addition to the reversible valve functions described with relatively small openings, these relatively large openings breaking open irreversibly and allowing a very rapid transfer of liquid only at or above a very high speed and/or only at or above a very large acting centripetal force.

[0039] FIG. 5 illustrates, in schematic section, a centrifuge cup 40 having a reversible valve function which is realized by an externally peripheral sealing ring 22 which, comparably with the embodiment in FIG. 3, closes openings within the wall of the centrifuge cup 40 in a manner dependent on centripetal force. In the wall of the centrifuge cup 40, additionally, there are further openings, preferably larger in diameter, which in this exemplary embodiment are closed by stoppers 45. These openings may alternatively be closed by adhesive tape, for example. The relatively large openings closed by stoppers 45 or other closure means break open irreversibly when the speed is appropriately high and discharge the liquid 1 from the centrifuge cup 40 very quickly. For the application, for example, of the recovery of washed cells from the centrifuge cup 40, this embodiment ensures that, during the very short time interval of the transfer of the detached cells from the centrifuge cup 40, the resuspended cells do not adhere again to the internal wall of the centrifuge cup 40, as would be the case in the event of prolonged spinning. The irreversible breaking-open of the valve function 45 by adhesive tape adhered externally to the centrifuge cup 40 in the region of the valve function 45, for example, can be achieved by the adhesive force failing at or above a certain magnitude of the acting centripetal force or at or above a certain threshold speed, and irreversibly exposing the openings. The closure may also be configured, for example, by means of a metal foil in such a way that the metal foil tears irreversibly above the aforementioned threshold speed. In a further embodiment, the openings may be closed with stoppers, plastic or metal stoppers for example, which shoot out when the centripetal force reaches a certain height or when the threshold speed is exceeded, and suddenly expose the openings. Through a combination of such irreversible valve functions with reversible valve functions, in particular, there are diverse possible uses for the centrifuge unit integrated into a microfluidic device. The combination shown in FIG. 5 of the reversible valve function from FIG. 3 and the irreversible valve function 45 is merely an example. In other embodiments, for example, the valve function 35 from FIG. 4 may be combined with an irreversible valve function.

[0040] Through targeted actuation of the centripetal force-dependent valve functions, by setting of the required speeds and also, optionally, by the use of selective lysis media and/or extraction media, it is possible to use the integrated centrifuge unit to carry out specific operations in the processing of the sample, such as, for example, the selective lysis of particular cells and/or, for example, the extraction of nucleic acids or the like.

[0041] A microfluidic device incorporating a centrifuge unit of this kind may be utilized, for example, for the removal of cellular material from whole blood and for recovery of the resultant cell-free blood plasma. From the cell-free blood plasma it is then possible, for example, for circulating cell-free tumoral DNA or other nucleic acids contained in the plasma to be isolated and analyzed. For the implementation of this processing, first of all a (whole) blood sample is input through a sample inlet directly into the centrifuge cup, using a pipette, for example. At a medium centrifuge speed, the cellular material (leucocytes, erythrocytes, circulating tumor cells, etc.) is first centrifuged over a sufficiently small time interval onto the internal wall of the centrifuge cup. Roughnesses or nanostructures or indentations made in a targeted way in the internal wall of the centrifuge cup here may further assist adhesion of the cells to the wall. The centrifuge speed is subsequently raised above a predeterminable threshold value, causing the triggering of the valve function in the manner described and spinning the cell-free blood plasma from the centrifuge cup, allowing it to be coupled into the fluidic network of the microfluidic device.

[0042] Another application example for a microfluidic device of this kind is the removal of erythrocytes and the purification and lysis of the remaining cells and also the extraction of the nucleic acids obtained from them. In this exemplary embodiment, following introduction, for example, of the whole blood sample into the centrifuge cup, a medium selective for erythrocyte lysis (an ammonium chloride solution, for example) is added in the required amount to the centrifuge cup. This erythrocyte lysis medium causes the red blood corpuscles to burst. After a sufficiently long waiting time, the centrifugation process is commenced at a medium speed, with the valve function still remaining closed. Leucocytes and any circulating tumor cells (CTCs) are sedimented onto the wall of the centrifuge cup, and surface structures on the internal wall of the centrifuge cup may reinforce the adhesion in the wall. After a sufficiently long centrifugation time, the speed is raised so that the valve function opens and the “supernatant” liquid, including the erythrocyte lysate, is spun out of the centrifuge cup and discarded. Then the sample input chamber and/or the centrifuge cup may be rinsed one or more times and freed from erythrocyte residues, for example. During rinsing, the cells, especially the leucocytes, remain adhering on the inner wall surface of the centrifuge cup, and so rinsing steps purify the cellular material on the wall of the centrifuge cup and remove impurities. Finally a lysis buffer is added to lyze the cell material on the wall of the centrifuge cup and the nucleic acids are released as a result. In the concluding centrifuging step, at high speed, the lysis medium with the nucleic acids contained therein is transferred out of the centrifuge cup into the sample input chamber and taken over into the fluidic network of the microfluidic device. As and when required, the media used may be temperature-conditioned, more particularly heated or cooled, and the lysis of the cells may be promoted by a heated lysis medium, for example.

[0043] In a further application example, erythrocytes may be removed from a whole blood sample and the remaining cells may be coupled into the fluidic network of the microfluidic device, where they are put to further use. After the input, for example, of a whole blood sample into the centrifuge cup, purification is commenced first by the metered addition of a lysis medium selective for erythrocyte lysis, ammonium chloride solution for example, in the desired amount into the centrifuge cup, by flooding, for example. Following the subsequent lysis of the erythrocytes, the centrifugation process is commenced at a medium speed, with leucocytes and any circulating tumor cells being sedimented onto the centrifuge cup wall, supported where appropriate by structurings on the inner wall surface of the centrifuge cup. After a sufficiently long centrifugation time, the speed is raised so that the valve function opens and the “supernatant” liquid is spun out of the centrifuge cup and discarded. The sample input chamber and/or the centrifuge cup are subsequently rinsed one or more times and in particular freed from erythrocyte residues. The remaining cellular constituents of the sample, freed from the erythrocytes, are present in the interior of the centrifuge cup and can be washed by the optional rinsing steps. Lastly a further medium is introduced in the desired amount into the centrifuge cup, in order to detach the cells present on the wall of the centrifuge cup. For this purpose it is possible to use, for example, an EDTA-containing medium. Detachment of the cells from the wall may be accomplished by leaving the cup to stand or may be assisted, for example, by slow rotation of the centrifuge cup with a periodic change in the direction of rotation. The rotational speed chosen here is usefully kept low enough that centripetal forces play no part in the sense of cell sedimentation, yet the liquid column keeps the entire internal wall of the centrifuge cup covered, and at the same time a transient flow is formed in the centrifuge cup. The eddying triggered by a change in the direction of rotation preferably facilitates the detachment of the cells from the inner wall of the centrifuge cup and so facilitates the transit of the cells into the liquid. In a last centrifuging step, preferably with a sharply commencing high speed, the detached cells within the medium are spun through the opened valves into the sample input chamber and transferred into the channel system of the microfluidic device. Provision may be made here for the sample material first to be collected, by appropriate connection of the channels in the microfluidic device, before the material enters into the actual channel system. In this application example as well, the temperature of the media used may be adjusted by preheating or precooling in order to support the processes.

[0044] The application examples for the integrated centrifuge unit, elucidated here with reference to whole blood samples, can also be applied to other sample liquids, and the centrifuge unit integrated into a microfluidic device may be utilized in principle for all microfluidic processes which rationally include a centrifuging step.