Sample carrier and rotation device

Abstract

The present invention relates to a sample carrier (1) for use in a rotation-based method for replicating or detecting DNA, comprising a disk-shaped substrate (2) and a number of cavities (4, 6, 8) formed in the substrate (2) for receiving a sample liquid, at least potentially containing DNA, during intended method steps (S1, ..., S4). The disk surface of the substrate (2) forms a heat input side (40), and the flat surface opposite the disk surface forms a heat output side. One cavity (6) or one of the cavities (4, 6, 8) is formed by an annular channel having first and second channel portions (50, 52) fluidly connected at both longitudinal ends by a connecting portion (54). The first channel portion (50) is offset in the thickness direction of the substrate (2) relative to the second channel portion (52).

Description

[Technical Field]

[0001] The present invention relates to a sample carrier for use in a method for replicating DNA, as well as to a rotating device configured and arranged for use in such a method, and also to the use of such a sample carrier and such a rotating device, respectively, in a method for replicating DNA. [Background technology]

[0002] DNA (deoxyribonucleic acid) is frequently analyzed for scientific genetic material analysis, paternity testing, etc., as well as for testing for existing diseases or for detecting pathogens. Today, this is relatively well known due to the spread of SARS-CoV-2 and the tests required to detect it. For this analysis (or detection), it is necessary to replicate specific segments of the DNA (and optionally also RNA) contained in a sample, for example, a mucosal tissue or blood sample. When RNA in a sample is detected or analyzed (e.g., for virus detection), it is first transcribed into DNA by so-called "reverse transcription" and then replicated.

[0003] To replicate DNA, the so-called polymerase chain reaction (abbreviated PCR) is typically used in a liquid reaction mixture. DNA is usually in the form of a double helix consisting of two complementary single strands of DNA. In PCR, the DNA is first separated into two single strands (the "denaturation step") by raising the temperature of the liquid reaction mixture, typically to between 90 and 96°C.

[0004] The temperature is then lowered again (the "annealing step," usually in the range of 50-70°C) to allow so-called primer molecules to specifically attach to the single strands. The primer molecules are complementary short DNA strands that bind to the single strands at defined positions. The primers serve as starting points for the enzymes known as polymerases, which fill in the existing single-stranded DNA with DNA building blocks (dNTPs) complementary to the sequence in the single strand in the so-called extension step. During this process, double-stranded DNA is again formed, starting from the primer molecules. Extension is usually carried out at the same temperature as the annealing step or at a slightly higher temperature, usually 65-75°C. After extension, the temperature is raised again for the denaturation step. The primer molecules, as well as the building blocks mentioned above, are also present in the reaction mixture. They are usually included in the starting mixture to which the sample is added.

[0005] This cycle of changing the temperature in the liquid reaction mixture through two or three temperature ranges is called PCR thermal cycling and is usually repeated 30 or 50 times. During each cycle, a specific DNA region is replicated. Generally, the thermal cycling of the liquid reaction mixture is reversed by controlling the external temperature within the reaction vessel. The reaction vessel is placed, for example, in a thermoblock, within which PCR thermal cycling is performed by heating and cooling a solid in thermal contact with the reaction vessel. In this way, heat is supplied to and removed from the liquid reaction mixture. Alternatively, heating and cooling concepts for PCR thermal cycling are temperature control of the fluid (particularly air and water) flowing around the reaction vessel, as well as radiation-based concepts using, for example, infrared or laser radiation.

[0006] In conventional polymerase chain reactions, the processing time is usually in the range of 45 minutes to 3 hours, and is therefore relatively time-consuming. Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to accelerate the overall course of the polymerase chain reaction, in particular the analysis. [Means for solving the problem]

[0008] This problem is solved according to the invention by a sample carrier having the features of claim 1. Furthermore, this problem is solved according to the invention by a rotation device having the features of claim 9. Furthermore, this problem is solved according to the invention by a method of use having the features of claim 15 and by a method of use having the features of claim 16. Further advantageous, and partly inventive in themselves, embodiments and further configurations of the invention are set out in the dependent claims and in the following description.

[0009] The sample carrier according to the invention and the rotating device according to the invention are preferably used together, but alternatively independently of each other (i.e., the sample carrier is used independently of the rotating device, or vice versa), in a method for DNA replication or verification ("detection"). According to this method, the sample carrier (or the sample carrier according to the invention), in particular at least one cavity of the sample carrier, is preferably first filled with a sample liquid that preferably or at least potentially contains DNA (e.g., in the case of pathogen testing). The sample carrier is then rotated around the rotation axis by the rotating device (or the rotating device according to the invention). The cavity, preferably the sample carrier, is then heated to a high temperature by the heating device on the heat input side lying in the rotation plane (i.e., in particular parallel to the rotation plane). Preferably, no heating is performed on the side opposite the heat input side. Due to the heating, convection of the sample liquid occurs in the cavity. Preferably, the convection occurs in a substantially circular fashion, with a first flow section extending, in particular, approximately parallel to the heat input side, a second flow section extending from the heat input side to the opposite heat release side (also referred to as the "cooling side"), a third flow section extending parallel to the heat release side, and a fourth flow section extending back (from the heat release side) to the heat input side. The sample liquid is thereby preferably guided through a denaturation zone (with a particularly high temperature value), a so-called annealing zone (also referred to as the primer hybridization zone), and an elongation zone, before returning to the denaturation zone. The circulation time of the sample liquid droplets along the convection flow path is then set (in particular "controlled"), in particular, by the rotation speed.

[0010] In particular, the circulation period of the liquid particles is also influenced by other parameters such as the shape of the cavity, the viscosity of the sample liquid, the density of the sample liquid, and the temperature gradient that occurs.

[0011] By heating the cavity from one side as described above, in other words, a temperature gradient (which therefore decreases from the heat input side to the heat output side) is applied to the sample liquid in the cavity, preferably perpendicular to the prevailing forces, in particular the centrifugal forces resulting from the rotation.

[0012] In particular, the fluid exchange between the denaturation zone and the annealing zone required for the polymerase chain reaction takes place through these flow sections, or flow sections, oriented perpendicular to the plane of rotation (i.e., the second and fourth flow sections).

[0013] Preferably, alongside the four flow sections there is a section of flow transverse to them, driven by centrifugal and / or Coriolis forces, which advantageously further promotes mixing of the sample liquid, thereby allowing as homogeneous a mixture as possible of the reaction partners (i.e. the DNA to be replicated, the primer molecules and the "strand members").

[0014] The term "circulation period" is understood here and below to mean the period (time) required for the (especially small) liquid particles to flow through the denaturation zone, the annealing zone (also called the primer hybridization zone), the elongation zone and back to the denaturation zone. The circulation period can be set in the range of 0.1 to 20 seconds depending on the number of rotations (and therefore the rotation speed). Within the cavity corresponding to the reaction chamber of the sample carrier, the average flow velocity can be set in the order of up to 22 mm / s.

[0015] Such short circulation times and / or high flow rates allow for particularly fast polymerase chain reactions, thereby advantageously saving processing time.

[0016] In a preferred variant of the method, the corresponding cavity is cooled on its heat release side (or also "cooling side") opposite the heat input side to a lower temperature value compared to the higher temperature value on the heat input side, which advantageously allows to regulate the temperature of the annealing zone (and optionally the elongation zone contained therein) and in particular to prevent further, or at least not negligible, heating of the sample liquid in the region of the annealing zone.

[0017] The sample carrier according to the invention is configured and arranged for use in the rotation-based method for replicating DNA as described above and below. The sample carrier comprises a disk-shaped substrate. Furthermore, the sample carrier comprises a number of preferably microfluidic cavities formed in the substrate, which accommodate a sample liquid therein that at least potentially (this is particularly the case in the case of an analysis for the presence of a pathogen) contains DNA (or, alternatively, RNA) during a intended method step. One flat surface (or: disk surface) of the substrate preferably forms the heat input side, and the flat surface (or: disk surface) opposite to the flat surface (i.e., the heat input side) particularly forms the heat release side (also referred to as the "cooling side"). One cavity, or possibly one of the cavities, is formed by an annular channel (i.e., preferably a loop-shaped or ring-shaped channel) having first and second channel portions. These two (i.e., first and second) channel portions are fluidly connected at least indirectly by connecting portions (or connecting channels) at both ends in the longitudinal direction. The first channel portion is arranged offset relative to the second channel portion in the thickness direction of the substrate (i.e., in particular, in the direction of the intended rotation axis). In other words, one of the two channel portions is offset toward the heat input side, and the other is offset toward the cooling side.

[0018] "Disc-shaped" is understood here and below in particular in the sense of "plate-shaped", i.e. in the sense that the corresponding body has a planar extent (preferably essentially independent of the geometric shape of its outer contour that defines the planar extent) that is many times greater than its thickness.

[0019] The term "a predetermined number" is understood here and below to mean, inter alia, the term "quantity," so that a predetermined number of elements refers both to a single element and to at least two elements.

[0020] "Microfluidic" is understood here and below to mean that at least one cavity has a dimension of less than 0.5 millimeters, or from 0.1 millimeters to 10-15 millimeters. In particular, at least one dimension, such as width or depth, is in the range of less than 0.5 millimeters. The longitudinal extension, in particular the longitudinal extension of the cavities forming the channels, can also exceed the aforementioned 15 millimeters.

[0021] Preferably, the annular channel forms a processing or reaction chamber in which polymerase chain reaction (abbreviated PCR) takes place during the intended use of the sample carrier. This is supported by the annular channel shape of the cavity. This is because, in particular, convection and gravity-driven flows are easily formed by the individual "liquid particles" flowing one after the other through the individual channel sections as described above. In particular, in the heated channel sections, upon rotation, the liquid particles can "rise" against the centrifugal force, while the cooled, and therefore denser or heavier, liquid particles in the other channel sections "sink" in the direction of the centrifugal force. The second and fourth flow sections mentioned above run through the connecting channel between the first and second channel sections. In particular, this improves the overall mixing and movement of the sample liquid contained in the annular channel, and therefore the processing. Furthermore, by offsetting the first and second channel portions toward the heat input and cooling sides, respectively (i.e., in the thickness direction), it is further advantageous that the heat input and heat release (i.e., cooling in particular) are primarily effective, and preferably limited to, the corresponding (i.e., nearby) channel portions. In other words, the cooling effect is reduced for channel portions offset toward the heat input side. The opposite is true for channel portions offset toward the cooling side.

[0022] Preferably, the first channel portion is arranged on the heat input side (i.e., towards the heat input side) and the second channel portion is arranged on the cooling side of the sample carrier (i.e., offset towards the cooling side). Preferably, in intended use, the first channel portion is used for heat input to the sample liquid and the second channel portion is correspondingly used for heat release. Further preferably, the first and second channel portions are also oriented parallel to the direction of centrifugal force (to be applied during the intended process) (in particular radially with respect to the axis of rotation about which the sample carrier rotates in intended use).

[0023] In a preferred embodiment, the first channel section (as described above) has a reduced cross-sectional area (at least in some areas) compared to the second channel section (positioned offset to the cooling side). The reduced cross-sectional area then leads, on the one hand, to an increased flow rate and, consequently, to a reduced residence time of the individual "liquid particles" in the first channel section. Furthermore, the surface area available for heat input is approximately smaller, thereby limiting the possible heat input.

[0024] In a further preferred embodiment, the first channel section, preferably arranged on the heat input side (i.e., offset toward the heat input side), is oriented toward the disk surface of the base body and has a reduced channel width (in addition to or as an alternative to a reduced cross-sectional area), particularly compared to the second channel section (at least in some areas) arranged on the cooling side. This makes the "active surface" for heat input smaller than the "active surface" for heat release. This allows the heat release to be matched to the heat input. In particular, this allows for particularly simple implementation of cooling via the surrounding atmosphere. Active, and therefore energy-consuming, cooling generation can be advantageously omitted. In contrast, active heating is required periodically anyway.

[0025] In particular, in embodiments in which the second channel section is offset toward the cooling side, the second channel section includes a cooling channel and an annealing channel, the annealing channel preferably connecting to the cooling channel in the intended flow direction of the sample liquid during processing and having a reduced channel width compared to the cooling channel. In this case, the cooling channel is used to cool the processing liquid as quickly as possible. Meanwhile, the cooling in the annealing channel is reduced, thereby ensuring that the temperature conditions therein are as constant as possible. Optionally, the cross-sectional areas of the cooling channel and the annealing channel are selected to be the same and / or the annealing channel is selected to have a greater "depth," i.e., a greater extension into the thickness direction of the substrate. In particular, in the former case, the flow rate also remains at least approximately the same. However, alternatively, the cross-sectional area of ​​the annealing channel is also reduced compared to the cooling channel, thereby increasing the flow rate therein and, consequently, shortening the residence time. Despite the names "cooling channel" and "annealing channel," accumulation of DNA building blocks can occur already in the cooling channel on DNA strands delivered from the first channel section and denatured there by heating.

[0026] In a preferred embodiment, the annealing channel alternatively has the same channel width as the cooling channel, but has a greater depth in contrast to the latter, thereby increasing the volume within the annealing channel compared to the cooling channel, thereby allowing a greater amount (specifically, a larger volume) of sample liquid to impede cooling, thereby slowing down cooling.

[0027] In one optional embodiment, the first channel section comprises two partial chambers (or "partial sections"): a "denaturation channel" and a "resistance channel." The resistance channel is then arranged "upstream," i.e., before the denaturation channel in the intended flow direction (and therefore, in particular, downstream of the annealing channel). Furthermore, the resistance channel is formed with a reduced width, preferably a reduced cross-sectional area, compared to the denaturation channel. This results in an acceleration of the sample liquid in the resistance channel. In particular, this resistance channel influences, on the one hand, the flow rate in the annealing channel and, on the other hand, the fluid resistance in the annular channel (for example, by more than 40%, preferably more than 50%) and thus the (at least theoretical) circulation time of the liquid particles through the respective channel section. The circulation time influences the amount of heat absorbed and released, and thereby the temperature values ​​generated in the sample liquid. Therefore, the resistance channel is advantageously also a "control element" in the design engineering for the respective temperature values ​​in the sample liquid.

[0028] Alternatively, the modification channel is omitted, and the modification can be carried out particularly upon suitable process implementation (e.g., external heating and / or relatively low rotation speeds), and in this case particularly in the first channel portion formed only as a resistive channel.

[0029] The above-mentioned shaping (or: "structuring") of the annular channel advantageously allows the setting (or: "control") of the circulation time and the individual temperature values ​​by the channel cross section or channel profile and / or by the rotation speed. In particular, the channel geometry can be adapted to the process parameters (e.g. heating and cooling temperature values) defined by the analytical device (in particular the rotating device described in more detail below) so that the time limitations are (no longer) defined primarily by the heating or cooling duration, but at least 20% or more by the biochemical process.

[0030] In one preferred embodiment, in addition to being offset in the thickness direction, the first and second channel portions are also offset from one another in the disk plane direction.

[0031] In a further preferred embodiment, the sample carrier comprises a thermal insulating layer, which is arranged below the second channel section in the direction towards the heat input side (or above, depending on the viewing direction; generally speaking, the thermal insulating layer is arranged between the second channel section and the heat input side) for at least part of its length. This advantageously prevents heat input from the heating chamber into the second channel section during intended operation (especially if it is offset in the thickness direction). Optionally, the thermal insulating layer is then assigned only to the cooling channel (e.g., arranged below), so that heat input into the cooling channel is prevented or at least reduced to a negligible level, and heat release is significantly dominant. Therefore, heat input into the subsequent annealing channel is "allowed" in this option, so that the sample liquid in this channel is cooled to a lesser extent or the temperature can be kept approximately constant (i.e., within a small temperature difference in °C, for example, within 10 °C or 5 °C or less).

[0032] In a preferred embodiment, the annular channel is connected to the bubble trap chamber in the inlet region where the annular channel is filled (especially with sample liquid) during intended use. Preferably, in this case, the gate connecting the bubble trap chamber to the annular channel is made thick enough to allow the passage of conventionally generated bubbles from the annular channel to the bubble trap chamber. For example, a gate thickness of at least 100 micrometers is sufficient to allow bubbles to pass into the bubble trap chamber, especially at a rotation speed of 20 Hz. Bubbles are generated, especially by heating the sample liquid. If bubbles remain in the annular channel, they can lead to blockages such as gas embolisms, especially in passages with small cross sections, especially in narrow slits. During intended use of the sample carrier in the above method, the annular channel is preferably filled with a large amount of sample liquid so that the sample liquid at least partially fills the bubble trap chamber. This further simplifies the flow of bubbles from the annular channel to the bubble trap chamber, since it is not necessary to overcome a liquid-gas interface. Furthermore, the bubble trap chamber is preferably positioned radially inward relative to the annular channel during intended rotation of the sample carrier, so that gas bubbles, which are lighter than the sample liquid, can "rise", i.e. move radially inward, against the gravitational field of the rotational drive.

[0033] Preferably, a bubble trap chamber is provided in each of the first and second channel portions, and more preferably extends radially.

[0034] In one optional embodiment, the annular channel has a third channel portion fluidly connected between the first and second channel portions, particularly downstream of the second channel portion. Preferably, this third channel portion is oriented (at least approximately) parallel to the first and second channel portions. However, in the thickness direction of the substrate of the sample carrier, the third channel portion is disposed between the first and second channel portions. This allows a temperature value to be established in the third channel portion during intended operation, preferably between the (average) temperature values ​​of the first and second channel portions. For example, when supporting DNA elongation, the target temperature value in the first channel portion is about 85-100°C, particularly about 95°C; in the second channel portion, about 50-75°C, preferably about 60°C; and in the third channel portion (if present), about 65-80°C, preferably about 72°C.

[0035] In a further optional embodiment, the sample body comprises a plurality of the above-mentioned annular channels, each of which has a different structure (i.e. preferably dimensions, in particular with regard to their cross section and width), whereby the residence times of the sample liquid in the individual regions are different, so that tests can be carried out in the sample carrier, in particular under constant heating and cooling conditions, upon different processing parameters (in particular different temperature values ​​and / or circulation times), optionally with different biochemistries.

[0036] The rotating device according to the present invention, which will be described in more detail below, is optionally an independent invention, and therefore an invention independent from the above-described sample carrier. Nevertheless, the use of the above-described sample carrier in the rotating device described here and below is particularly advantageous. The rotating device according to the present invention is configured and provided for use in the above-described rotation-based method. To this end, the rotating device comprises an analysis chamber and a sample holder arranged in the analysis chamber. The latter is for mounting at least one sample carrier, in particular the above-described sample carrier having (or a) a predetermined number of cavities formed in a substrate, which in a intended method step contain a sample liquid, at least potentially containing DNA. Furthermore, the rotating device comprises a rotation drive for rotating the sample holder about a rotation axis during intended operation. Furthermore, the rotating device comprises a heating device for thermoregulating the atmosphere in a partial region of the analysis chamber forming a heating chamber to a target heating temperature during intended operation, and a cooling device for thermoregulating the atmosphere in a partial region of the analysis chamber forming a cooling chamber to a target cooling temperature during intended operation. The heating chamber and the cooling chamber are fluidically separated from each other by the sample holder, at least in cooperation with the sample carrier mounted therein. Furthermore, the rotation device comprises a controller (also referred to as "control device"), which is coupled in control technology to the rotation drive and the heating device as well as the cooling device and is configured to set the rotation speed of the sample holder as well as the target heating and cooling temperatures.

[0037] Preferably, the heating and cooling are therefore effected via the temperature-controlled atmosphere of the heating or cooling chamber, respectively. Particularly preferably, the respective atmosphere is air, which results in a simple construction of the rotating device.

[0038] In a preferred embodiment, the controller is formed, at least at its core, by a microcontroller having a processor and a data memory, in which the functions for carrying out the method according to the invention are programmatically implemented in the form of operating software (firmware). When the operating software is executed in the microcontroller, the method is thereby executed automatically, if necessary in interaction with an operator. However, within the scope of the present invention, the controller can alternatively also be formed by non-programmable electronic components, for example an ASIC, in which the functions for carrying out the method are implemented by circuit-technical means.

[0039] In a preferred embodiment, the rotating device comprises a housing enclosing the analysis chamber, and thus both the heating and cooling chambers. In other words, the housing does not divide the analysis chamber. The division into the heating and cooling chambers is rather performed by the sample holder or sample carrier. The sample holder, or the sample carrier attached thereto in the intended operation, then forms a sealing gap with the wall of the housing, in particular with the side wall of the housing. The sealing gap is calculated so as to reduce or inhibit gas exchange between the heating and cooling chambers. For example, the sealing gap has a width (i.e., the distance between the sample holder or sample carrier and the side wall) of 1 mm or less, preferably less than 1 mm, in particular 0.5 mm or less.

[0040] A further advantageous feature for the sealing effect between the heating chamber and the cooling chamber is that the housing wall, together with the sample holder or sample carrier, forms a kind of labyrinth seal between the heating chamber and the cooling chamber. A labyrinth seal usually has a relatively high sealing effect in a contactless sealing concept. In this case, the housing wall, in particular the side wall, preferably has a circumferential groove into which the sample holder or sample carrier engages. Again, the sealing gap preferably has a dimension of 1 mm or less.

[0041] Preferably, the analysis chamber is cylindrical. The sample holder, either alone or together with at least one or more sample carriers attached to the sample holder, forms a circular plate that resembles the analysis chamber. The sealing gap is thereby preferably the same around the entire circumference. Preferably, in the case of a labyrinth seal, the housing can be hinged open or disassembled to load the sample holder and, optionally, for maintenance. The release surface of the housing is then advantageously located in the groove.

[0042] In a further preferred embodiment, the sample holder of the rotation device is configured to receive the sample carrier on the heat input side (here of the sample holder) facing the heating chamber. This is particularly true when the rotation drive is arranged in the area of ​​the cooling chamber. However, it is also possible, alternatively, to arrange the rotation drive on the side of the heating chamber, so that the sample holder receives the sample carrier on the cooling side (of the sample holder) facing the cooling chamber. In any case, the sample holder has at least one window connecting the heat input side and the cooling side (of the sample holder). Through this window, the area of ​​a certain number of cavities of the sample carrier to be cooled or heated (especially of the first or second channel part) is in heat transfer connection with the corresponding cooling or heating chamber during intended operation. That is, the area of ​​the sample carrier to be cooled (especially the second channel part) is in connection with the cooling chamber when the sample carrier is located on the heat input side of the sample holder (and therefore in the heating chamber). In the case of the above-described sample carrier according to the invention, its channel part (in particular the second channel part) offset to the cooling side optionally projects into or through the window towards the cooling side, this also applies to the region to be heated if the sample carrier is arranged on the cooling side of the sample holder.

[0043] In an optional embodiment, preferably when the sample carrier according to the invention is used with a rotation device, the sample holder has a thermal insulation layer, which is arranged so that at least a part of the area to be cooled and / or heated of a predetermined number of cavities of the sample carrier is shielded from the temperature influence of the heating or cooling chamber during the intended operation. This is particularly true if the sample carrier itself does not have a thermal insulation layer. The thermal insulation layer of the sample holder is optionally formed by an element arranged separately on the sample holder, for example by a material with low thermal conductivity. The thermal insulation layer of the sample holder serves the same purpose as the thermal insulation layer of the sample carrier according to the invention described above.

[0044] In a preferred embodiment, the cooling device of the rotating device has a controllable valve for connecting the cooling chamber to the surroundings of the rotating device and / or a fan for (particularly actively) introducing ambient air, preferably ambient air, into the cooling chamber. This makes it possible to dispense with active cooling, such as by using a type of air conditioning system (i.e., active cooling). This is particularly advantageous in that this approach (controllable valve or fan) is technically simple to implement. The target cooling temperature in the cooling chamber is set by the controller to approximately 50°C (i.e., with a deviation of, for example, ±5°C). This relatively high temperature value compared to the normal ambient temperature results from (optionally intentional) leakage through the above-mentioned seal gap and / or heat conduction through the sample holder. Preferably, a temperature sensor is arranged in the cooling chamber and connected to the controller to regulate the temperature to this temperature value. The controller opens one or more valves in the event of a temperature rise, thereby allowing exchange with the surroundings. If necessary and if present, the controller operates the fan to transport more ambient atmosphere, particularly air, throughout the cooling chamber and thereby enhance the cooling effect.

[0045] Preferably, the controller is configured to control the heating device so that the temperature value in the heating chamber is approximately 80-120°C. Preferably, a temperature sensor is also arranged in the heating chamber for this purpose. The heating device optionally comprises a heater wire, a surface heater, etc. In intended operation, the sample holder rotates together with the respective sample carriers mounted thereon, advantageously swirling the atmosphere in the heating chamber and thereby homogenizing the temperature. Furthermore, the relative movement of the sample carriers with respect to the atmosphere improves convective heat transfer, in particular because a stationary insulating boundary layer between the sample carriers and the heating chamber is constantly eliminated or not formed.

[0046] According to the present invention, the above-described sample carrier according to the present invention is used in the method described at the beginning. The sample carrier is then first filled with a sample liquid, at least potentially containing DNA, and then rotated around its axis of rotation by a rotating device, optionally the above-described rotating device according to the present invention. At least the first channel part is then at least partially heated to a high temperature by the atmosphere, the temperature of which is regulated by the heating device, thereby generating a convection current of the sample liquid in the annular channel of the corresponding cavity. As an alternative to the rotating device according to the present invention, a contact or surface heater, preferably integrated into the sample holder, is optionally used instead of the above-described heating device for regulating the temperature of the atmosphere in the heating chamber. In this case, the first (or heated) channel part is heated on one side by thermal conduction, for example by a Peltier element or a resistive heating element.

[0047] Furthermore, according to the present invention, the above-described rotation device according to the present invention is used in the method described at the beginning. Optionally, sample carriers other than the above-described sample carrier according to the present invention can also be used. However, preferably, a sample carrier according to the present invention is used. Within the scope of the method, at least a portion of one cavity of the sample carrier, or possibly at least a portion of one of the cavities, is at least partially heated to a high temperature by air temperature-regulated by a heating device, and preferably another portion (preferably of the same cavity) is cooled by air preferably at a lower temperature present in a cooling chamber. Due to the heating, in particular the temperature difference caused by the additional cooling, convection of the sample liquid is generated in the corresponding cavity.

[0048] Hereinafter, embodiments of the present invention will be described in more detail with reference to the drawings. [Brief explanation of the drawings]

[0049] [Figure 1] 2 shows a schematic view of the underside of a sample carrier with a predetermined number of cavities; FIG. [Figure 2] 1 is a schematic, semi-transparent side view of an embodiment of a rotation device for use in the present method. [Figure 3] 10 is a schematic, semi-transparent side view of another embodiment of a rotation device for use in the present method. [Figure 4] 1 is a schematic flow chart of a method for replicating DNA. [Figure 5] FIG. 2 shows a schematic detail view from below of an embodiment of a cavity of a sample carrier; [Figure 6] 2 shows a schematic side view of an embodiment of a cavity of a sample carrier in a detailed view; FIG. [Figure 7] 3 shows a schematic detail view from below of another embodiment of a cavity of a sample carrier; FIG. [Figure 8] 4 shows a schematic side view of another embodiment of a cavity of a sample carrier in a detailed view; FIG. [Figure 9]3 shows a schematic detail view from below of another embodiment of a cavity of a sample carrier; FIG. [Figure 10] 3 shows a schematic side view of an alternative embodiment of the cavity of the sample carrier in a detailed view; FIG. DETAILED DESCRIPTION OF THE INVENTION

[0050] Corresponding parts are always provided with the same reference numbers in all the figures.

[0051] 1 shows, in a broad, schematic view, a sample carrier 1 configured and arranged for use in a rotation-based method for replicating or detecting DNA, which will be described in more detail below with reference to FIG. 4. The sample carrier 1 comprises a disk-shaped, i.e., flat, in this example semicircular, substrate 2 in which a plurality of microfluidic cavities are formed, shown here by way of example only: a filling chamber 4 into which an acquired sample can be introduced, a processing chamber 6 arranged "downstream" thereof, and a connecting channel 8 therebetween. The size of the processing chamber 6 relative to the substrate 2 is shown greatly exaggerated here in order to illustrate features that will be described in more detail below.

[0052] 2 and 3 show two embodiments of a rotation device 10 configured and arranged for use, preferably with a sample carrier 1, in a rotation-based method for replicating DNA. The rotation device 10 comprises a housing 12, which by its side walls 14 encloses a cylindrical housing interior (hereinafter referred to as "analysis chamber 16"). Furthermore, the rotation device 10 comprises a sample holder 18, on which the sample carrier 1 is mounted when the method is performed (i.e., in intended operation). The sample holder 18 can be rotated about a rotation axis 22 by a rotation drive 20. The sample holder 18 is therefore a turntable.

[0053] The sample holder 18 is arranged to divide the analysis chamber 16 into two halves. The upper part in FIGS. 2 and 3 forms a heating chamber 24. The rotation device 10 is equipped with a heating device 26 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. For temperature control, the rotation device 10 is equipped with a cooling device 30. In the illustrated embodiment, the cooling device includes a fan 32, which, in intended operation, causes the cooling chamber 28 to be circulated by a cooling air flow formed by sucked in outside air. Furthermore, the cooling device 30 includes a controllable valve 34, through which air can be exhausted from the cooling chamber 28 to the ambient environment or can be admitted to the cooling chamber without the fan 32 being activated.

[0054] There is a controller for the rotary device 10, not shown in detail, for controlling the rotary drive device 20, the heating device 26, and the cooling device 30 (ie, the fan 32 and the valve 34).

[0055] To keep the transfer of hot air from the heating chamber 24 to the cooling chamber 28 to a minimum, the seal gap 36 between the sidewall 14 and the sample holder 18 is kept to 1 mm or less.

[0056] In a further embodiment, the housing 12 is configured to be openable by means of a hinge 38 between the heating chamber 24 and the cooling chamber 28. This allows for easy installation of the sample holder 18 and / or maintenance of the rotation device 10. The outer edge of the sample holder 18 fits into a groove 39 cut into the side wall 14, thereby forming a labyrinth seal (see FIG. 3). In principle, the housing 12 of the embodiment according to FIG. 2 can also be hinged open in a similar manner to allow installation of the sample holder 18, but not necessarily in the plane of the sample holder 18.

[0057] In a further embodiment, not shown, an automatic feeding of the sample carrier 1 into the rotating device 10 (comparable to a CD or DVD drive) is provided.

[0058] Furthermore, in a preferred embodiment, the rotating device 10 is equipped with a code reader for reading, for example, barcodes and / or QR codes, so that the detailed analytical results for the current sample can be transferred to a database via a network.

[0059] To replicate DNA, in the first method step S1 (see FIG. 4), a sample carrier 1 and a DNA-containing sample are prepared. The sample solution is generated after the sample is introduced into the loading chamber 4. In addition to the DNA to be replicated, it also contains primer molecules, deoxynucleoside triphosphates ("dNTPs"), which are the building blocks for forming new DNA strands, as well as a polymerase and its cofactors. Furthermore, the liquid is buffered. Preferably, a liquid is pre-stored in the loading chamber 4 or in a separate chamber (not shown) to "wash" the sample material from the sample carrier (e.g., a cotton swab) and to serve as a carrier liquid for the above-mentioned reagents. Optionally, some of these reagents are also initially added in the form of (dried) substances pre-stored in the processing chamber 6. In the second method step S2, the loaded sample carrier 1 is placed on the sample holder 18 and fixed there. The sample carrier 1 is then placed on the heat input side 40 of the sample holder 18, which is located in the heating chamber 24.

[0060] In a third method step S3, the air in the heating chamber 24 is thermostated by the heating device 26 to approximately 100°C, which is the high temperature value in the described method. In parallel with the thermostation, the rotation drive 20 drives the sample holder 18 to rotate about the rotation axis 22, whereby each cavity of the sample carrier 1 also rotates about the rotation axis 22. The cooling device 30 thermostates the air in the cooling chamber 28 to a low temperature value of approximately 50°C. The rotation of the sample holder 18 causes a movement and therefore a mixing of the air in the heating chamber 24 as well as the air in the cooling chamber 28.

[0061] As can be seen from FIGS. 1 and 5 , the processing chamber 6 of the sample carrier 1 has an annular channel structure, which is formed, in turn, by a first channel portion 50 and a second channel portion 52. These channel portions 50 and 52 are elongated and extend parallel to each other (at least approximately, i.e., optionally with a slight, single-digit angle offset) and parallel to (at least approximately parallel to) a radial line (which, in the intended operating state, is perpendicular to the rotation axis 22). In other words, the two channel portions 50 and 52 are oriented in the direction of centrifugal force during processing during the intended rotation. The channel portions 50 and 52 are fluidly connected at their ends by a connecting channel 54. Furthermore, the channel portions 50 and 52 are offset from each other in the thickness direction of the substrate 2, i.e., in the direction of the rotation axis 22. Specifically, the first channel portion 50 is offset towards the heat source in the intended use state of the sample carrier 1, i.e. towards the heating chamber 24 in the rotation device 10 of this embodiment. Conversely, the second channel portion 52 is offset towards the cooling chamber 28. To enable heat exchange between the air in the cooling chamber 28 and the processing chamber 6, at least with the second channel portion 52, the sample holder 18 has a window 56 through which air can flow from the cooling chamber 28 into the second channel portion 52. Optionally, the second channel portion 52 protrudes beyond the plane of the heat input side 40 of the sample holder 18, thus into the window 56, or protrudes downwards, i.e. beyond the sample holder 18 into the cooling chamber 28 (not shown).

[0062] Thereby, in method step S3, due to its greater "proximity" (in relation to the second channel portion 52) to the heating chamber 24, relatively more heat is introduced into the first channel portion 50 than into the second channel portion 52. The rotation of the sample holder 18 and the resulting relative movement with respect to the air also supports convective heat exchange between the two channel portions 50 and 52 together with the heating chamber 24 or the cooling chamber 28.

[0063] Heating of the first channel portion 50 by the heating chamber 24 and cooling of the second channel portion 52 by the cooling chamber 28 creates a temperature gradient in the channel structure of the processing chamber 6 that extends parallel to the rotation axis 22. Due to the rotation, an artificial gravity field is created in the radial direction relative to the rotation axis 22. Furthermore, the temperature gradient creates a density difference in the sample liquid.

[0064] This temperature-induced density difference, combined with the artificial gravity field, results in buoyancy-driven convection, the primary flow direction of which is essentially radial due to the artificial gravity field. In other words, the primary buoyancy component is directed radially inward. Due to the annular structure of the processing chamber 6, liquid elements flow radially inward due to their heating and associated density decrease in the first channel portion 50. Correspondingly, liquid elements flow radially outward due to gravity due to cooling and associated density increase in the second channel portion 52. Because the two channel portions 50 and 52 are connected to form a ring, fluid elements flow radially inward from the first channel portion 50 through the connecting channel 54 to the second channel portion 52, and then correspondingly return to the first channel portion 50 at its end. However, due to the presence of rotational centrifugal force (to the right in FIG. 5 ) and rotational Coriolis force, there is also (homogeneous) mixing of the sample fluid across the basic flow path of the convection current. The convection current speed increases with increasing rotation speed.

[0065] As can be seen from Figures 5 and 6, the second channel section 52 has two sub-chambers or "sub-chambers," the radially inner one of which is called the "cooling channel 58" and the radially outer one connected to it is called the "annealing channel 60." The cooling channel 58 has a larger width in the planar direction of the substrate 2 than the annealing channel 60, thereby enabling the fastest possible cooling to the "annealing temperature" of approximately 65°C. In this embodiment, the cross section of the annealing channel 60 is also selected to be smaller than the cross section of the cooling channel 58, thereby achieving a relatively high outflow velocity. This results in less heat dissipation and less heat loss during the transition to the first channel section 50.

[0066] The first channel part 50 likewise has two partial chambers, the radially outer one of which is called the resistance channel 62 and the radially inner one of which is called the denaturation channel 64. The resistance channel 62 has a further reduced cross section compared to the annealing channel 60 and the connecting channel 54, thereby accelerating the sample liquid and controlling (or setting) the flow through the annealing channel 60. In the denaturation channel 64, its (in this example) again enlarged cross section allows the temperature to be kept at least approximately constant (for example, between 90 and 100°C, in particular about 95°C).

[0067] 7 and 8 show a further embodiment of the processing chamber 6. The difference with the previous embodiment lies in the dimensions of the annealing channel 60 relative to the cooling channel 58, as well as the shape of the first channel section 50. The annealing channel 60 has the same "depth" or "height" (i.e., the dimension extending in the direction of the rotation axis 22) as the cooling channel 58. This results in less flow acceleration compared to the embodiment according to FIGS. 5 and 6. The first channel section 50 is formed almost identically over its entire length. No distinction is made in this embodiment between the resistance channel 62 and the denaturation channel 64. The first channel section 50 is relatively elongated and has a tapered middle section, formed almost nozzle-like. Denaturation also occurs here in the tapered central section as soon as the corresponding temperature is reached. This is possible in the exemplary embodiment (at least in the case of a rotary device with a contact heater). The cross-sectional area of ​​the first channel section 50 (its tapered region) is then 0.162 mm . 2 and the second channel portion 52 is formed in such a way that, at a rotation speed of 10 Hz of the sample carrier 1, the sample liquid remains in the first channel portion 50 long enough to reach the denaturation temperature value. For higher rotation speeds, the cross-sectional area of ​​the first channel portion 50 can be reduced accordingly for the higher flow rates that are then achieved.

[0068] To reduce the effect of the heated air (or another heating medium) in the heating chamber 24 on the second channel portion 52, an insulating layer 66 is disposed therebelow. For example, the insulating layer may be a gas-filled "cushion," such as a hollow or foam plate.

[0069] 9 and 10 show a further embodiment of the processing chamber 6. In this embodiment, the annealing channels 60 are narrower but deeper than the cooling channels 58. This increases the volume within the annealing channels 60, resulting in low heat losses, even though in this embodiment the insulating layer 66 is only located below the cooling channels 58. The denaturing channels 64 (which are clearly present in this embodiment) are formed with an enlarged cross section compared to the resistance channels 62, similar to the embodiment according to FIGS. 5 and 6.

[0070] In each of the above-described embodiments, the first channel section 50 and the second channel section 52 are offset from one another in the tangential direction, which on the one hand simplifies the intermediate arrangement of the heat insulating layer 66. On the other hand, however, it also makes it possible to monitor the progress in the two channel sections 50 and 52, for example by means of a fluorescence detector, especially if the substrate 2 is made transparent at least in the region of the processing chamber 6.

[0071] Furthermore, in each of the above-described embodiments, the two channel sections 50 and 52 each have an inlet 68 (or "inlet region") through which the sample liquid is filled. The inlet 68 has two inlet chambers, also called "bubble traps 70," each of which is fluidly connected to one of the two channel sections 50 and 52 via a gate 72. The amount of sample liquid supplied is selected so that after the channel sections 50 and 52 are filled as intended, i.e., when sample liquid is present in both channel sections 50 and 52 and the connecting channel 54, some of the sample liquid is still present in the bubble trap 70. The gate 72 is sized so that gas bubbles formed in the intended operation by heating the sample liquid can "rise" through the liquid against the artificial gravity field to the bubble trap 70 and collect there without "clogging" the gate. This is facilitated by the partially filled bubble trap 70.

[0072] In this case, the dimensions of the channel sections 50 and 52 and the connecting channel 54 are selected so that, at a rotation speed in the range of 5 to 40 Hz, the sample liquid in the annealing channel 60 has a temperature value of approximately 65°C, and in the first channel section 50, the sample liquid has a temperature value above the melting temperature of DNA, specifically above 90°C, and especially about 90°C.

[0073] In particular, the method steps S1 to S3 can also be performed at least partially simultaneously with one another. In particular, the sample holder 18 does not have to be stationary during the filling of the processing chamber 6. Similarly, the heating device 26 can also heat the air already in the heating chamber 24.

[0074] Optionally, in one embodiment of the method, method step S3 is maintained for a predetermined time, after which, in a fourth method step S4, rotation of the sample holder 10 and heating by the heating device 26 are stopped. Optionally, the fourth method step S4 can also be initiated if a sufficiently high conversion of the reagent is detected by the above-mentioned fluorescence detector.

[0075] It should be noted that the subject matter of the present invention is not limited to the examples described above, but rather further embodiments of the invention can be derived by those skilled in the art from the above description, and in particular the individual features of the invention and of the variant embodiments described by the various examples can also be combined in other ways. [Explanation of symbols]

[0076] 1 sample carrier 2 Base 4 Filling chamber 6 Processing Chamber 8 connection channels 10 Rotating device 12 Housing 14 Side wall 16 Analysis Chamber 18 Sample holder 20 Rotational drive unit 22 Rotation axis 24 Heating Chamber 26 Heating device 28 Cooling Chamber 30 Cooling device 32 fans 34 Valve 36 Seal gap 38 Hinge 39 Groove 40 Heat input side 50 Channel Section 52 Channel section 54 connection channels 56 Windows 58 Cooling Channels 60 Annealing Channels 62 Resistance Channel 64 Denaturing Channels 66 Insulation Layer 68 Inlet 70 Bubble Trap Gate 72 S1~S4 Method steps

Claims

1. A sample carrier (1) for use in a rotation-based method for replicating or detecting DNA, comprising: a disk-shaped substrate (2) that rotates around a rotation axis (22) parallel to the thickness direction; a predetermined number of cavities (4, 6, 8) formed in the substrate (2) for accommodating a sample liquid at least potentially containing DNA in steps (S1, ..., S4) of the method; The disk surface of the base (2) forms a heat input side (40), and the flat surface opposite to the disk surface forms a heat release side; the cavity (6) or one of the cavities (4, 6, 8) is formed by an annular channel having a first channel portion (50) and a second channel portion (52) fluidly connected at both longitudinal ends by a connecting portion (54), the first channel portion (50) and the second channel portion (52) extending radially relative to the rotation axis (22); The sample carrier (1) is arranged such that the first channel portion (50) is offset relative to the second channel portion (52) in the thickness direction of the substrate (2), so that the first channel portion (50) is arranged on the heat input side (40) and the second channel portion (52) is arranged on the heat release side.

2. 2. The sample carrier (1) according to claim 1, wherein the first channel portion (50) has a reduced cross-sectional area compared to the second channel portion (52).

3. 3. The sample carrier (1) according to claim 1 or 2, wherein the first channel portion (50) has a reduced channel width in the direction of the disk surface of the substrate (2) compared to the second channel portion (52).

4. 4. The sample carrier (1) of claim 3, wherein the second channel portion (52) includes a cooling channel (58) and an annealing channel (60) connected to the cooling channel and formed with an increased depth compared to the cooling channel (58).

5. A sample carrier (1) according to any one of claims 2 to 4, wherein the first channel portion (50) comprises a modification channel (64) and a resistance channel (62) preceding the modification channel and formed with a reduced width compared to the modification channel (64).

6. The sample carrier (1) according to any one of claims 1 to 5, wherein the first channel portion (50) and the second channel portion (52) are offset from one another in the direction of the disk surface.

7. A sample carrier (1) according to any one of claims 1 to 6, comprising a heat insulating layer (66) arranged underneath the second channel portion (52) in the direction towards the heat input side for at least part of its length.

8. the annular channel is connected to a bubble trap chamber (70) at an inlet region (68) where filling of the annular channel occurs in intended use; A sample carrier (1) according to any one of claims 1 to 7, wherein the gate (72) connecting the bubble trap chamber (70) to the annular channel has a thickness large enough to allow the passage of conventionally occurring air bubbles from the annular channel to the bubble trap chamber (70).

9. A rotation device (10) for use in a rotation-based method for DNA replication or detection, comprising: an analysis chamber (16); a sample holder (18) arranged in the analysis chamber (16) for mounting at least one sample carrier (1) according to any one of claims 1 to 8, which sample carrier has a predetermined number of cavities (4, 6, 8) formed in a substrate (2) for receiving a sample liquid at least potentially containing DNA during the steps of the method, a rotation drive (20) for rotating the sample holder (18) about an axis of rotation (22) in intended operation; a heating device (26) for thermoregulating the atmosphere in a sub-region of said analysis chamber (16) that forms a heating chamber (24) in intended operation to a target heating temperature; a cooling device (30) for thermoregulating the atmosphere in a partial region of the analysis chamber (16) that forms a cooling chamber (28) in intended operation to a target cooling temperature, wherein the heating chamber (24) and the cooling chamber (28) are fluidically separated from each other by the sample holder (18), at least in cooperation with the sample carrier (1) attached thereto; a controller coupled to the rotation drive device (20), the heating device (26), and the cooling device (30) in terms of control technology and configured to set the rotation speed of the sample holder (18), as well as the target heating temperature and the target cooling temperature.

10. a housing (12) enclosing both the heating chamber (24) and the cooling chamber (28); 10. The rotating device (10) of claim 9, wherein the sample holder (18) or the sample carrier (1) attached thereto during intended operation forms a sealing gap (36) together with the housing wall (14) of the housing (12), the sealing gap being formed to reduce gas exchange between the heating chamber (24) and the cooling chamber (28).

11. The housing wall (14) together with the sample holder (18) or the sample carrier (1) is adapted to fit into a groove (39) formed in the circumferential direction of the housing wall (14) so ​​as to form a space between the heating chamber (2) and the sample holder (18) or the sample carrier (1).

11. The rotating device (10) of claim 10, wherein a kind of labyrinth seal is formed between the rotor (4) and the cooling chamber (28).

12. the sample holder (18) is configured to receive the sample carrier (1) on a heat input side (40) facing the heating chamber (24) or on a cooling side facing the cooling chamber (28); 12. The rotating device (10) according to claim 9, wherein the sample holder (18) has at least one window (56) connecting the heat input side (40) and the cooling side to each other, via which window the area of ​​the predetermined number of cavities (4, 6, 8) of the sample carrier (1) to be cooled or heated is connected in a heat transfer technique to the cooling chamber (28) or the heating chamber (24) respectively in the intended operation.

13. 13. The rotating device (10) of claim 12, further comprising a thermal insulating layer arranged such that at least a portion of the area to be cooled and / or heated of the predetermined number of cavities (4, 6, 8) of the sample carrier (1) is insulated from the temperature influence of the heating chamber (24) or the cooling chamber (28) during intended operation.

14. The rotating device (10) according to any one of claims 9 to 13, wherein the cooling device (30) comprises a controllable valve (34) for connecting the cooling chamber (28) to the surroundings of the rotating device (10) and / or a fan (32) for drawing ambient air into the cooling chamber (28).

15. Use of a sample carrier (1) according to any one of claims 1 to 8 in a method for the replication or detection of DNA, comprising: The sample carrier (1), which contains a sample liquid at least potentially containing DNA, is rotated about a rotation axis (22) by a rotation device (10) according to any one of claims 9 to 14, heating at least the first channel portion (50) at least partially to an elevated temperature value by a temperature-controlled environment via a heating device (26); A method of using a sample carrier (1), wherein heating induces a convection current of said sample liquid in the annular channel of the corresponding cavity (6).

16. Use of a rotating device (10) according to any one of claims 9 to 13 in a method for DNA replication or detection, comprising: A sample carrier (1) according to any one of claims 1 to 8, which has a predetermined number of cavities (4, 6, 8), and in at least one of said cavities contains a sample liquid which at least potentially contains DNA, is rotated around a rotation axis (22) by the rotating device (10), heating at least a portion of said one cavity (6) or at least a portion of one of said cavities (4, 6, 8) to a high temperature value at least partially by atmospheric air temperature-regulated by a heating device (26); A method of using a rotating device (10) for generating convection in the sample liquid in the corresponding cavity (6) by heating.

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