SAMPLE CARRIER, ROTATION APPARATUS AND METHODS OF USING THE SAMPLE CARRIER AND ROTATION APPARATUS

Abstract

A sample carrier is used in a rotation-based method for reproducing or detecting DNA. The sample carrier has a disc-like main part and a plurality of cavities formed in the main part, in which cavities, a sample fluid at least potentially containing DNA is received. A disc side of the main part forms a heat entry side and the flat side facing away therefrom forms a heat discharge side. The cavity or one of a plurality of cavities, as applicable, is formed by an annular channel having a first and a second channel portion, which are fluidically connected at both longitudinal ends by a connection portion in each case. The first channel portion is arranged offset relative to the second channel portion in the thickness direction of the main part.

Claims

1. A sample carrier for use in a rotation-based method for amplification or detection of deoxyribonucleic acid (DNA), the sample carrier comprising: a disk-shaped base body having a plurality of cavities formed therein, in which, a sample liquid that at least potentially contains the DNA is received, said disk-shaped base body further containing: a disk side forming a heat input side; a flat side facing away from said disk side and forming a heat output side; at least one of said cavities is formed by an annular channel with a first and a second channel section which are fluidically connected at both longitudinal ends by means of a respective connection section; and said first channel section disposed offset, in a thickness direction of said disk-shaped base body, with respect to said second channel section.

2. The sample carrier according claim 1, wherein said first channel section is disposed on said heat input side and has a reduced cross-sectional area compared to said second channel section disposed on said heat output side.

3. The sample carrier according to claim 1, wherein said first channel section is disposed on said heat input side and, compared to said second channel section disposed on said heat output side, has a reduced channel width oriented in a disk surface direction of said disk-shaped base body.

4. The sample carrier according to claim 3, wherein said second channel section includes a cooling channel and, adjoining said cooling channel, an annealing channel formed with an increased depth compared to said cooling channel.

5. The sample carrier according to claim 2, wherein said first channel section has a denaturation channel and, in front of said denaturation channel, a resistance channel formed with a reduced width compared to said denaturation channel.

6. The sample carrier according to claim 1, wherein said first and said second channel section are offset from each other in a disk surface direction.

7. The sample carrier according to claim 1, further comprising a thermal insulation layer which is disposed underneath said second channel section over at least part of its length in a direction of said heat input side.

8. The sample carrier according to claim 1, further comprising a bubble trap chamber having in an inlet region, wherein said annular channel is connected to said bubble trap chamber via said inlet region through which said annular channel is filled during an intended use.

9. The sample carrier according to claim 8, wherein said inlet region has a gate, which connects said bubble trap chamber to said annular channel, and has a thickness that gas bubbles which normally occur are able to pass through from said annular channel into said bubble trap chamber.

10. A rotation device for use in a rotation-based method for amplification or detection of deoxyribonucleic acid (DNA), the rotation device comprising: an analysis chamber; at least one sample carrier having a base body with a plurality of cavities formed in said base body, in which, in an intended operation, a sample liquid that at least potentially contains the DNA is received; a sample holder disposed in said analysis chamber for holding said at least one sample carrier; a rotary drive by means of which said sample holder is rotated about an axis of rotation during the intended operation; a heating device by means of which an atmosphere in a subregion of said analysis chamber forming a heating chamber is controlled to a target heating temperature during the intended operation; a cooling device by means of which, during the intended operation, an atmosphere in a subregion of said analysis chamber forming a cooling chamber is controlled to a target cooling temperature, wherein said heating chamber and said cooling chamber are fluidically separated from each other by said sample holder, at least in cooperation with said sample carrier held thereon; and a controller which is linked in terms of control technology to said rotary drive, said heating device and said cooling device and is configured to specify a speed of rotation of said sample holder and also the target heating temperature and the target cooling temperature.

11. The rotation device according to claim 9, further comprising a housing with a housing wall jointly enclosing said heating chamber and said cooling chamber, wherein said sample holder, or said at least one sample carrier held thereon during the intended operation, forms a sealing gap with said housing wall of said housing, said sealing gap is configured to reduce a gas exchange between said heating chamber and said cooling chamber.

12. The rotation device according to claim 11, wherein said housing wall forms, with said sample holder or said at least one sample carrier, a labyrinth seal between said heating chamber and said cooling chamber.

13. The rotation device according to claim 10, wherein said sample holder is configured to receive said at least one sample carrier on a heat input side facing said heating chamber or on a cooling side facing said cooling chamber, and wherein said sample holder has at least one window connecting said heat input side and said cooling side to each other, through which said at least one window a region of said plurality of cavities of said at least one sample carrier that is to be cooled or heated is accordingly connected, during the intended operation, to said cooling chamber or said heating chamber so as to permit heat transfer.

14. The rotation device according to claim 13, further comprising a thermal insulation layer disposed such that at least part of a region of said plurality of cavities of said at least one sample carrier that is to be cooled and/or heated is shielded, during the intended operation, from a temperature control effect of said heating chamber or said cooling chamber.

15. The rotation device according to claim 10, wherein said cooling device has: a controllable valve for connecting said cooling chamber to an environment of the rotation device; and/or a fan for flooding said cooling chamber with ambient atmosphere.

16. The rotation device according to claim 12, wherein said housing wall has a groove formed circumferentially therein, wherein said labyrinth seal between said heating chamber and said cooling chamber is formed by said sample holder or said at least one sample carrier engaging in said groove formed circumferentially in said housing wall.

17. A method for amplification or detection of deoxyribonucleic acid (DNA), which comprises the steps of: providing a sample carrier according to claim 1; receiving the sample carrier, in which the sample liquid that at least potentially contains the DNA in a rotation device, and rotating the sample carrier about an axis of rotation by means of the rotation device; heating at least the first channel section to a given temperature value, at least in some sections, by means of an atmosphere that is temperature-controlled by means of a heating device of the rotation device; and generating, on account of the heating, a convection flow of the sample liquid within the annular channel of one of the cavities.

18. A method for amplification or detection of deoxyribonucleic acid (DNA), which comprises the steps of: providing the rotation device according to claim 10; rotating the at least one sample carrier having the plurality of cavities, in at least one of the cavities the sample liquid at least potentially containing the DNA is received, about an axis of rotation by means of the rotation device; heating at least one section of the cavity or several of the cavities to a given temperature value, at least in some sections, by means of an atmosphere that is temperature-controlled by means of the heating device; and generating, on account of the heating, a convection flow of the sample liquid within the cavity.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0052] FIG. 1 is a schematic view of an underside of a sample carrier with a number of cavities;

[0053] FIGS. 2 and 3 are schematic and quasi-transparent side views each showing an alternative exemplary embodiment of a rotation device used in a method;

[0054] FIG. 4 is a flowchart illustrating a method for amplification of DNA; and

[0055] FIGS. 5-10 are schematic detailed views of an underside and of a side, different exemplary embodiments of a cavity of the sample carrier.

DETAILED DESCRIPTION OF THE INVENTION

[0056] Corresponding parts are always provided with the same reference signs in all of the figures.

[0057] Referring now to the figures of the drawings in detail and first, particularly to FIG. 1 thereof, there is shown a sample carrier 1 in a roughly schematic manner, which is configured and provided for use in a rotation-based method for amplification or detection of DNA, described in more detail below with reference to FIG. 4. The sample carrier 1 has a disc-shaped, i.e. flat, base body 2 which is semicircular in the present exemplary embodiment. Several microfluidic cavities are formed in the base body 2, of which FIG. 1 shows merely by way of example a filling chamber 4, into which a sample taken can be introduced, a process chamber 6 arranged “downstream” thereof, and a connection channel 8 between these two. The size of the process chamber 6 in relation to the base body 2 is shown here in a greatly exaggerated manner in order to illustrate the properties described in more detail below.

[0058] FIGS. 2 and 3 show two exemplary embodiments of a rotation device 10 which is likewise configured and provided for use in a rotation-based method for amplification of DNA, preferably together with the sample carrier 1. The rotation device 10 has a housing 12 which, with its side wall 14, encloses a circular cylindrical housing interior, referred to below as “analysis chamber 16”. Furthermore, the rotation device 10 has a sample holder 18. The sample carrier 1 is mounted on the latter when the method is being carried out (i.e. during the intended operation). The sample holder 18 can be rotated about an axis of rotation 22 by means of a rotary drive 20. Thus, the sample holder 18 is a turntable.

[0059] The sample holder 18 is arranged in the analysis chamber 16 in such a way that it divides the latter into two parts. The upper part in Figs 2 and 3 forms a heating chamber 24. The rotation device 10 has a heating device 26 which is configured to heat the atmosphere, specifically the air in the heating chamber 24. The lower part of the analysis chamber 16 in FIGS. 2 and 3 forms a cooling chamber 28. The rotation device 10 has a cooling device 30 for its temperature control. In the exemplary embodiment shown, there is shown a fan 32 by means of which, during the intended operation, a flow of cooling air, formed by air sucked in from outside, flows through the cooling chamber 28. In addition, the cooling device 30 contains a controllable valve 34 through which air can be discharged from the cooling chamber 28 into the environment or can be admitted without the fan 32 being activated.

[0060] A controller of the rotation device 10 for controlling the rotary drive 20, the heating device 26 and the cooling device 30, i.e. the fan 32 and the valve 34, is present but not shown in any detail.

[0061] In order to keep the passage of warm air from the heating chamber 24 into the cooling chamber 28 as small as possible, a sealing gap 36 between the side wall 14 and the sample holder 18 is kept at less than 1 mm.

[0062] In a further exemplary embodiment, the housing 12 can be folded open up by means of a joint 38 between the heating chamber 24 and the cooling chamber 28. As a result, the sample holder 18 can be easily loaded and/or the rotation device 10 serviced. The outer edge of the sample holder 18 lies in a groove 39 which is worked into the side wall 14. This creates a labyrinth seal (see FIG. 3). In principle, the housing 12 of the exemplary embodiment according to FIG. 2 can also be folded open in order to be able to load the sample holder 18, but not necessarily in the plane of the sample holder 18.

[0063] In a further exemplary embodiment, which is not shown, the sample carrier 1 is automatically drawn into the rotation device 10, comparable to a CD or DVD drive.

[0064] Furthermore, in an expedient exemplary embodiment, the rotation device 10 has a code reader for reading in, for example, barcodes and/or QR codes, by means of which an analysis result for the current sample can be forwarded in a specified manner to a database via a network.

[0065] For the amplification of DNA, the sample carrier 1 and the sample containing DNA are made available in a first method step S1 (see FIG. 4). The sample liquid forms after the sample has been introduced into the filling chamber 4 and, in addition to the DNA to be amplified, it also contains primer molecules, deoxynucleoside triphosphates (“dNTPs”), structural building blocks for the formation of new DNA strands, and also polymerase and co-factors of the polymerase. In addition, the liquid is buffered. A liquid is preferably stored in the filling chamber 4 or in another chamber (not shown) and is used to “wash out” the sample material from a sample carrier (e.g. a swab) and as a carrier liquid for the abovementioned reagents. Optionally, some of these reagents are also only added in the form of upstream (dry) substances in the process chamber 6. In a second method step S2, the filled sample carrier 1 is placed onto the sample holder 18 and fastened to it. The sample carrier 1 rests on a heat input side 40 of the sample holder 18 located in the heating chamber 24.

[0066] In a third method step S3, the air in the heating chamber 24 is controlled to about 100 degrees Celsius by means of the heating device 26. In the method described, this represents a high temperature value. In parallel with this, the rotary drive 20 drives the sample holder 18 to rotate about the axis of rotation 22, such that each cavity of the sample carrier 1 is also rotated about the axis of rotation 22. The air in the cooling chamber 28 is controlled to a low temperature value of approximately 50 degrees Celsius by means of the cooling device 30. As a result of the rotation of the sample holder 18, there is also a movement and therefore mixing of the air in the heating chamber 24 and in the cooling chamber 28.

[0067] As can be seen from FIGS. 1 and 5, the process chamber 6 of the sample carrier 1 has a channel structure which runs in a ring shape and is in turn formed by a first channel section 50 and a second channel section 52. These channel sections 50 and 52 are elongate and run (at least approximately, i.e. optionally with an angular offset of a few, single-figure angular degrees) parallel to each other and (at least approximately parallel) to a radial which, in the intended operating state, is perpendicular to the axis of rotation 22. In other words, during the method, the two channel sections 50 and 52 are aligned in the direction of the centrifugal force during the intended rotation. The channel sections 50 and 52 are each fluidically connected at the ends by connection channels 54. In addition, the channel sections 50 and 52 are offset from each other in the direction of thickness of the base body 2, i.e. in the direction of the axis of rotation 22. Specifically, the first channel section 50 is offset toward a heat source in the normal state of use of the sample carrier 1, i.e. toward the heating chamber 24 in the present exemplary embodiment of the rotation device 10. Conversely, the second channel section 52 is offset toward the cooling chamber 28. In order to enable heat exchange between the air in the cooling chamber 28 and the process chamber 6, at least with the second channel section 52, the sample holder 18 has a window 56 through which air can flow from the cooling chamber 28 to the second channel section 52. Optionally, the second channel section 52 protrudes beyond the level of the heat input side 40 of the sample holder 18 and thus lies in the window 56 or even protrudes to the underside, i.e. into the cooling chamber 28 beyond the sample holder 18 (not shown).

[0068] Thus, in method step S3, comparatively more heat is introduced into the first channel section 50, on account of its greater “closeness” to the heating chamber 24 (as seen in relation to the second channel section 52) than into the second channel section 52. On account of the rotation of the sample holder 18 and the resulting relative movement to the air, the convective heat exchange of the two channel sections 50 and 52 with the heating chamber 24 and the cooling chamber 28 is also supported.

[0069] As a result of the heating of the first channel section 50 from the heating chamber 24 and the cooling of the second channel section 52 from the cooling chamber 28, a temperature gradient that runs parallel to the axis of rotation 22 forms within the channel structure of the process chamber 6. As a result of the rotation, an artificial gravitational field forms radially with respect to the axis of rotation 22. Furthermore, the temperature gradient leads to differences in density in the sample liquid. These temperature-related density differences, in conjunction with the artificial gravitational field, lead to a buoyancy-driven convection flow, the main flow direction of which is fundamentally radial on account of the artificial gravitational field. In other words, the main buoyancy component is directed radially inward. On account of the annular structure of the process chamber 6, liquid elements flow radially inward as a result of their heating in the first channel section 50 and the associated decrease in density. Correspondingly, as a result of the cooling in the second channel section 52 and the associated increase in density, liquid elements flow radially outward under the force of gravity. Since the two channel sections 50 and 52 are connected to form a ring, the liquid elements flow radially inward from the first channel section 50 through the connection channel 54 into the second channel section 52 and, at the end thereof, back into the first channel section 50. However, on account of the centrifugal forces of the rotation (directed to the right in FIG. 4) and the Coriolis force that is also present due to the rotation, there is also a (homogeneous) mixing of the sample liquid transversely with respect to the basic flow path of the convection flow. The speed of the convection flow increases as the speed of rotation increases.

[0070] As can be seen from FIGS. 5 and 6, the second channel section 52 has two sub-chambers, of which the radially inner one is referred to as the “cooling channel 58” and the one connected to it radially to the outside as the “annealing channel 60”. The cooling channel 58 has a greater width than the annealing channel 60 in the direction of the plane of the base body 2, thus permitting the fastest possible cooling to an “annealing temperature” of about 65 degrees Celsius. In this exemplary embodiment, the cross section of the annealing channel 60 is chosen to be smaller than that of the cooling channel 58, thereby permitting a comparatively higher outflow speed and thus a reduced heat dissipation, and also a lower heat loss at the transition to the first channel section 50.

[0071] The first channel section 50 also has two sub-chambers, of which the radially outer one is referred to as the resistance channel 62 and the radially inner one as the denaturation channel 64. The resistance channel 62 has a cross section that is further reduced in relation to the annealing channel 60 and also to the connection channel 54. As a result, the sample liquid is accelerated, and the flow through the annealing channel 60 is also controlled (or also predetermined). In the denaturation channel 64, the temperature (for example from 90 to 100, in particular about 95 degrees Celsius) can be kept at least approximately constant on account of the enlarged cross section of said channel in the present exemplary embodiment.

[0072] A further exemplary embodiment of the process chamber 6 is shown in FIGS. 7 and 8. The differences from the previous exemplary embodiment lie in the dimensions of the annealing channel 60 in relation to the cooling channel 58 and in the design of the first channel section 50. The annealing channel 60 has the same “depth” or “height” (i.e. the dimension in the direction of the axis of rotation 22) as the cooling channel 58. As a result, the flow is accelerated less than in the exemplary embodiment of FIGS. 5 and 6. The first channel section 50 is designed to be almost conformal over its entire length. A distinction between resistance channel 62 and denaturation channel 64 is not made here. The first channel section 50 is designed in the manner of a nozzle with a comparatively elongate, tapered central part. Denaturation also takes place here in the tapered central part as soon as the appropriate temperature is reached. This is possible in an exemplary embodiment, at least in the case of a rotation device with contact heating, in which the cross-sectional area of the first channel section 50 (in its tapered region) is 0.162 mm.sup.2 and the second channel section 52 is designed in such a way that, at a rotational speed of 10 Hz of the sample carrier 1, the sample liquid remains in the first channel section 50 for such a time that the denaturation temperature value is reached. For higher speeds, the cross-sectional area of the first channel section 50 can be correspondingly reduced on account of the then higher flow rate.

[0073] In order to reduce the effect of the heated air of the heating chamber 24, or of another heating means, on the second channel section 52, the latter is underlaid with a thermal insulation layer 66. For example, the thermal insulation layer is a gas-filled “cushion”, e.g. a hollow or foamed plate.

[0074] FIGS. 9 and 10 show a further exemplary embodiment of the process chamber 6. In this case, the annealing channel 60 is narrower but deeper than the cooling channel 58. As a result, the volume in the annealing channel 60 is increased, so that the heat loss can be kept low, although the thermal insulation layer 66 here is only placed underneath the cooling channel 58. Comparable to the exemplary embodiment according to FIGS. 5 and 6, the denaturation channel 64, once again of pronounced extent here, is designed with an enlarged cross section in relation to the resistance channel 62.

[0075] In each of the above-described exemplary embodiments, the first and second channel sections 50 and 52 are offset from each other in a tangential direction. On the one hand, this simplifies the intermediate storage of the thermal insulation layer 66, but on the other hand it also makes it possible, particularly in the case of the base body 2 being designed to be transparent at least in the region of the process chamber 6, to monitor the processes within the two channel sections 50 and 52, e.g. by means of a fluorescence detector or the like.

[0076] In addition, in each of the exemplary embodiments described above, the two channel sections 50 and 52 are assigned an inlet 68 (or also “inlet region”), via which the filling with the sample liquid takes place. This inlet 68 has two inlet chambers, also referred to as “bubble traps 70”, each of which is fluidically connected to one of the two channel sections 50 and 52 via a gate 72. The amount of sample liquid supplied is selected in such a way that, after channel sections 50 and 52 have been filled as intended, i.e. when there is sample liquid in both channel sections 50 and 52 and in the connection channels 54, there is also some sample liquid in the bubble traps 70. The gates 72 are dimensioned in such a way that gas bubbles, which form during normal operation on account of the heating of the sample liquid, can “rise” through the gates counter to the artificial gravitational field into the bubble traps 70 and can collect there without “clogging” the gates. This is favored by the partially filled bubble traps 70.

[0077] The dimensions of the channel sections 50 and 52 and of the connection channels 54 are chosen in such a way that, at rotational speeds in the range of 5 to 40 Hz, the sample liquid in the annealing chamber 60 has a temperature value of about 65 degrees Celsius and, in the first channel section 50, has a temperature value above the melting temperature of the DNA, specifically above 90 degrees Celsius, in particular around 90 degrees Celsius.

[0078] In particular, method steps S1 to S3 can also take place at least partially at the same time. In particular, the sample holder 10 does not have to stand still while the process chamber 6 is being filled. Similarly, the heating device 26 can already heat the air in the heating chamber 24.

[0079] In an optional embodiment of the method, method step S3 is maintained for a specified duration. Then, in a fourth method step S4, the rotation of the sample holder 10 and the heating by means of the heating device 26 are stopped. Optionally, the fourth method step S4 can also be initiated if a sufficiently high conversion of reagents is detected by means of the abovementioned fluorescence detector.

[0080] The subject matter of the invention is not restricted to the exemplary embodiments described above. Rather, further embodiments of the invention can be derived from the above description by a person skilled in the art. In particular, the individual features of the invention that have been described with reference to the various exemplary embodiments, and the design variants thereof, can also be combined with one another in a different way.

[0081] The following is a summary list of reference numerals and the corresponding structure used in the above description of the invention.

LIST OF REFERENCE SIGNS

[0082] 1 sample carrier
2 base body
4 filling chamber
6 process chamber
8 connection channel
10 rotation device
12 housing
14 side wall
16 analysis chamber
18 sample holder
20 rotary drive
22 axis of rotation
24 heating chamber
26 heating device
28 cooling chamber
30 cooling device
32 fan
34 valve
36 sealing gap
38 joint
39 groove
40 heat input side
50 channel section
52 channel section
54 connection channel
56 window
58 cooling channel
60 annealing channel
62 resistance channel
64 denaturation channel
66 thermal insulation layer
68 inflow
70 bubble trap
72 gate
S1-S4 method step