Cartridge for rotation-based analytical method with single-sided heat input, rotation-based analytical method, and method of using the cartridge

Abstract

The present invention relates to a cartridge (1) for a rotation-based analytical method using one-sided heat input. The cartridge (1) comprises a planar substrate (2) having a microfluidic channel and chamber structure (4) formed therein, with a plurality of processing chambers interconnected by the channels, a plurality of positioning and / or fixing elements (32) formed on the substrate (2) for positioning and / or fixing the substrate (2) to a support plate (34) of an analytical device for performing the analytical method, and a cover body (14) fixed to the substrate (2) and disposed on one side of an upper side (16) of the substrate (2) opposite the heat input side (8) and covering at least one chamber (56).

Description

[Technical Field]

[0001] The present invention relates to a cartridge for a rotation-based analytical method with one-sided heat input.The invention also relates to a rotation-based analytical method.Furthermore, the invention also relates to the use of a cartridge according to the invention. [Background technology]

[0002] Rotation-based analytical methods are used in the medical field, particularly with the use of so-called cartridges with microfluidic channel and chamber structures. They are primarily used for the detection of genetic material, primarily in the form of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), for the analysis of existing diseases or, in general, for the detection of pathogens, as well as for scientific genetic material analysis. For this purpose, it is necessary to replicate specific segments of the genetic material (DNA or RNA) contained in a sample, such as a mucosal tissue or blood sample. When RNA is detected or analyzed in a sample (such as for the detection of viruses), 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. Primer molecules are short, complementary DNA strands that bind to the single strands at defined positions. The primer molecules (also called "primers" for short) serve as starting points for enzymes called polymerases, which fill in the existing single-stranded DNA sequence with complementary building blocks (dNTPs) in the so-called extension step. In 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.

[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 typically repeated 30 or 50 times. During each cycle, a specific DNA region is replicated. Generally, the thermal cycle 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 the PCR thermal cycle is reversed by heating and cooling a solid in thermal contact with the reaction vessel, with heat being supplied to and removed from the liquid. Alternatively, heating and cooling concepts for reversing the PCR thermal cycle include temperature control of the fluid (especially air and water) flowing around the reaction vessel, as well as radiation-based concepts using heat supply, for example, ultraviolet or laser irradiation. In rotation-based methods, the reaction vessel is, for example, a chamber within the cartridge described above, which is heated accordingly. Furthermore, a cartridge typically shaped like a disk is rotated. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to further improve the rotation-based analysis method. [Means for solving the problem]

[0007] This problem is solved according to the invention by a cartridge having the features of claim 1. Furthermore, this problem is solved by an analysis method having the features of claim 9. Furthermore, this problem is solved according to the invention by the use of a cartridge having the features of claim 11. Advantageous, partly inventive, embodiments and further developments of the invention are set out in the dependent claims and the following description.

[0008] The cartridge according to the present invention is configured and provided for use in a rotation-based analytical method with one-sided heat input. For this purpose, the cartridge comprises a planar, i.e., particularly substantially two-dimensional, substrate in which a microfluidic channel and chamber structure is formed and in which a plurality of processing chambers are interconnected by the channels. In particular, during the analytical method, a liquid to be analyzed is transported between these processing chambers via at least one channel. Furthermore, the cartridge comprises a plurality of positioning and / or fixing elements formed on the substrate for positioning and / or fixing the substrate to a support plate of an analytical device configured and provided for carrying out the analytical method. Furthermore, the cartridge comprises a cover body fixed to the substrate, which is arranged on one side of the substrate opposite the heat input side and covers at least one (processing) chamber of the channel and chamber structure.

[0009] "Substantially two-dimensional" is understood here and below to mean, in particular, that the dimensions in the two-dimensional plane are many times, preferably five times or more, larger than any (thickness) variations transverse to this plane. The terms "microfluidic" or "microfluidic channel and chamber structure" are understood here and below to mean, in particular, that the dimensions of at least one direction, in particular the depth or width, of the components, preferably at least the channels, are at least predominantly in the range of 30 to 700 micrometers. In the case of channels, they are preferably of this order in both the depth and width directions. Some chambers may then have larger dimensions.

[0010] In particular, in rotation-based analytical methods primarily involving one-sided heat input, the cover body can prevent relatively large heat losses on the side opposite (above) the heat input side, particularly due to convection. This maintains a relatively uniform temperature distribution within the chamber, which is beneficial for the reaction process within the chamber. Furthermore, especially in the case of one-sided heat input, the heated boundary layer on the heat input side of the chamber is constantly "rinsed" by the moving liquid, resulting in the heated partial volume being mixed with the remaining liquid. This also promotes relatively rapid denaturation of the genetic material strands. Apart from uniform temperature distribution, such mixing also enables other uniform reaction conditions. This is because reaction partners, particularly specific additives (so-called "PCR primers" in the case of polymerase chain reaction, or "PCR"), are regularly pre-stored in the specific chamber where the reaction is performed, favoring their dissolution and mixing. Therefore, products or intermediate products generated locally during the reaction can also be advantageously mixed uniformly with other reaction partners. Furthermore, a particularly uniform temperature distribution (especially in the case of PCR) advantageously allows for relatively specific (i.e., particularly accurate or "correct") primer hybridization (also called "primer annealing") throughout the chamber. The addition of primers to the "correct" DNA (target) sequence is, as is known, temperature-dependent, with the closer the current temperature value is to the melting point of the DNA junction, the more specific the addition to the target sequence. Thus, when the temperature value in the chamber varies significantly, non-specific primer hybridization occurs regularly in the "cooler" regions, while more specific primer hybridization occurs in the hotter regions of the chamber.

[0011] In a preferred embodiment, the chamber covered by the cover body is an amplification chamber for the replication of genetic material, in particular a so-called pre-amplification chamber, in which the genetic material contained in the sample is replicated in order to make available in subsequent method steps a sufficient amount of genetic material for various test methods or for statistically sufficiently reliable tests.

[0012] The cartridge is then preferably configured such that the channel and chamber structure as a closed system has a plurality of successive chambers connected by channels in which the sample material is processed, prepared for testing, and tested, which has the advantage that potentially contaminated test objects, such as genetic material, do not have to be removed from the system.

[0013] For (pre)amplification of genetic material, temperature treatment is preferably performed according to the PCR thermal cycle described above. The genetic material is thereby heated and cooled to multiple different target temperatures in the corresponding chambers. Rotation of the cartridge generates an artificial gravitational field within the chamber, acting radially relative to the axis of rotation. One-sided heat input generates a rotating flow within the chamber. The flow moves from the heated chamber side toward the upper side of the substrate, approximately perpendicular to the radial direction, and then, due to increased density due to cooling, moves radially outward along the upper side (thus toward "artificial gravity") and then back toward the heat input side. Furthermore, the additional Coriolis force adds a steady force component to the rotating flow associated with this heat input, which in turn favors mixing within the chamber. This results in homogenization of the temperature and the above-mentioned reactants (i.e., sample materials, reaction partners, (intermediate) products, etc.). The cover reduces heat dissipation to the upper side, resulting in a uniform temperature within the chamber. Furthermore, the temperature of the heating element allows the temperature in the chamber to be set or controlled relatively stably and accurately (by mixing, advantageously relatively quickly), which, as mentioned above, is particularly advantageous during PCR, preferably during the so-called annealing stage (especially during primer hybridization), since this allows as uniform and specific binding of the primers as possible.

[0014] In a preferred embodiment, the cover body at least nearly completely (i.e., nearly, for example, within a maximum of 10 percent deviation) covers the upper side of the base, thereby enabling the above-described effect to be achieved in other chambers as well. In an optional variation, the cover body has an opening or transparent window through which test results from one or more "read chambers" can be read. Optionally, in the latter case, the opening is covered by a transparent sticker, however, the surface of the sticker spanning the opening is preferably adhesive-free.

[0015] In an advantageous embodiment, the cover body has a frame web that protrudes toward the upper side of the substrate and surrounds the covered chamber. This prevents or significantly reduces convection currents parallel to the surface of the substrate, particularly air currents flowing between the surface of the substrate and the cover body, at least throughout the covered chamber, preferably the (pre-)amplification chamber. This allows particularly high temperature uniformity (or, in other words, particularly small temperature deviations) to be achieved within the chamber (in a "quasi-steady" state, especially after relatively long holding times of the process parameters, i.e., heating temperature and rotation speed, for example, 30 seconds or more). In particular, temperature differences of less than 10 Kelvin, preferably less than 5 Kelvin, and especially about 2 Kelvin, can be achieved.

[0016] Preferably, the frame web rests on the substrate in the intended use state. Alternatively, the frame web is positioned at a small distance from the substrate (in the intended use state), in particular at a distance of less than 0.5 millimeters, preferably less than about 0.1 millimeters (i.e., 0.1 millimeters or less).

[0017] In particular, the area of ​​the covered chamber is selected to be relatively large (especially compared to other chambers and / or channels) to further enhance the possible temperature change rate of the covered chamber. For example, the covered chamber may be 2 x 5 mm in size with a "depth" of about 1.1 mm. 2 ~11×16mm 2 This allows a large surface area to be available for heat input compared to the typical dimensions of microfluidic channel and chamber structures, as is well known, and therefore allows for relatively large rates of temperature change.

[0018] In a further preferred embodiment, the covered chamber protrudes upward above the base body, i.e., the chamber forms a raised portion, in particular a stepped plateau (i.e., with right-angled or at least approximately right-angled side walls), on the upper side of the base body, with the chamber, in particular the plateau, and in particular its side walls (which in particular also form at least part of the side walls of the chamber), then partially overlapping the frame web.

[0019] In one preferred variant, the frame web is made from the same material as the cover body and in particular integrally (i.e. monolithically) with the cover body. Alternatively, for example when the frame web is arranged on the base and it is desired to achieve particularly effective airtightness of the volume above the amplification chamber (i.e. in the space enclosed by the base and the cover body with the frame web), the frame web is produced from a flexible material, for example a thermoplastic elastomer, for example by a two-component injection molding process.

[0020] mosquito The bar body is made of, in particular, thermoplastic plastic and is preferably manufactured by injection molding, which allows for a particularly economical production of the cartridge.

[0021] At that time, the base of the cartridge is ,blood It is formed from a substrate, particularly a thermoplastic substrate, into which the channel and chamber structure is molded, and a sealing coating, particularly a sealing film, which is firmly bonded to the substrate after the sealing step, thereby sealing the channel and chamber structure.

[0022] The rotation-based analytical method according to the present invention uses the cartridge described above. The cartridge is first fixed to a support plate of an analytical device, in particular a type of turntable. The support plate is then rotated while holding the cartridge. In other words, the cartridge preferably rotates in a plane parallel to its upper surface. Depending on the respective method step, heat is introduced into the base of the cartridge on one side (preferably locally limited to a single chamber or only a part of a chamber) by a predetermined number (preferably multiple) of heating elements arranged on the support plate. Furthermore, depending on the respective method step, the rotation speed is varied between a low rotation speed range of up to 20 Hz, a medium rotation speed range of 20-40 Hz, and a high rotation speed range of 40 Hz or higher.

[0023] By means of the heat input and / or different rotation speeds it is thereby possible to control how the sample material and / or the liquid containing the analyte is transported through the channel and chamber system or how it is influenced in the respective chamber in the respective method step. "Influence" of the sample material is understood here and below to mean in particular different "treatments", for example mechanical treatments or the triggering of biochemical reactions, mixing or the measurement of a liquid amount for a subsequent step.

[0024] "Analyte" is understood here and below to mean, in particular, a material (particularly a molecule) that supports a reaction, preferably DNA or RNA, such as a specific enzyme, amino acid, protein, or a material that supports downstream analysis of a reaction, for example, a molecule that causes a specific luminescence or fluorescence of a given reaction product. Such an analyte is preferably pre-stored in (part of) a significant number of these chambers, especially if several chambers are present. For example, the above-mentioned additives are such analytes.

[0025] Because the method uses the cartridge described above, the method also has all the advantages of the cartridge equally.

[0026] In a preferred variant of the method, PCR is carried out in a chamber covered by the cover body, i.e., in particular, in an amplification chamber. To this end, the liquid temperature in the chamber is set to 50-75°C on the one hand and 80-100°C on the other hand by cyclic heat input. That is, in the first cycle step, one temperature range is set first, and in the second cycle step, the corresponding other temperature range is set. Preferably, a medium to high rotation speed (i.e., at least 20 Hz, preferably 40 Hz or higher) is used.

[0027] According to the use according to the invention, the cartridge described here and in more detail below is used in a spin-based analytical method.

[0028] Hereinafter, embodiments of the present invention will be described in more detail with reference to the drawings. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic exploded view of a cartridge for use in a rotation-based analytical method. [Figure 2] FIG. 2 is a schematic plan view showing the heat input side of the base of the cartridge. [Figure 3] 1 is a schematic plan view of the support plate of an analytical device in which the cartridge is intended to be used; FIG. [Figure 4] 1 is a schematic side view of the support plate of an analytical device in which the cartridge is intended to be used; [Figure 5] 4 is a view according to FIG. 3 of the support plate with the partially assembled cartridge. [Figure 6] 5 is a view according to FIG. 4 of the support plate with the cartridge in its intended use state. [Figure 7] FIG. 10 is a schematic view showing the underside of the cartridge cover body. [Figure 8] 3 is a view according to FIG. 2 of the base of the cartridge for explaining the partial steps of the analysis method. [Figure 9] 3 is a view according to FIG. 2 of the base of the cartridge for explaining the partial steps of the analysis method. [Figure 10] 3 is a view according to FIG. 2 of the base of the cartridge for explaining the partial steps of the analysis method. [Figure 11] 3 is a view according to FIG. 2 of the base of the cartridge for explaining the partial steps of the analysis method. [Figure 12] FIG. 2 is a schematic detail view showing a cross section of a chamber of the cartridge. [Figure 13] FIG. 2 is a schematic partial cross-sectional view of a chamber of the cartridge. [Figure 14] FIG. 2 is a schematic partial cross-sectional view of a chamber of the cartridge. [Figure 15] FIG. 2 is a schematic partial cross-sectional view of a chamber of the cartridge. [Figure 16] FIG. 3 is a view according to FIG. 2 showing the base of the cartridge to explain further partial steps of the analysis method. [Figure 17] FIG. 3 is a view according to FIG. 2 showing the base of the cartridge to explain further partial steps of the analysis method. [Figure 18] FIG. 3 is a view according to FIG. 2 showing the base of the cartridge to explain further partial steps of the analysis method. [Figure 19] FIG. 3 is a view according to FIG. 2 showing the base of the cartridge to explain further partial steps of the analysis method. DETAILED DESCRIPTION OF THE INVENTION

[0030] Corresponding parts are always provided with the same reference numbers in all the figures.

[0031] FIG. 1 shows a schematic diagram of a sample container called a "cartridge" (or, due to its planar shape resembling half a disk, also referred to as "disk 1" for short). This disk 1 is used in a spin-based analytical method, which will be described in detail later. The disk 1 comprises a substrate 2 (also referred to as a "substrate") having a microfluidic channel and chamber structure 4. This channel and chamber structure 4 also has a plurality of chambers, which will be described in detail later, each connected to another by assigned channels (see FIG. 2). In the unassembled state, the chambers and channels form "open" basin-like recesses and groove-like recesses, respectively, in the substrate 2. Therefore, the disk 1 also comprises a sealing film 6 (or "sealing coating"), which is heat-sealed onto the microfluidic substrate 2 to seal the channel and chamber structure 4 from the side referred to as the "heat input side 8" hereinafter. The substrate 2 has a lateral access 10 to the channel and chamber structure 4, through which sample material can be introduced into the channel and chamber structure 4. The access portion 10 can be reversibly closed by a capsule 12, allowing the insertion of sample material and subsequent resealing. The disk 1 also comprises a cover body (hereinafter referred to as "cover 14" for short) that rests on the "upper side 16" of the substrate 2 (or the "underside" relative to the heat input side 8), and in this embodiment, the cover body is fixed to the substrate 2 by engaging hooks 18 (see FIG. 7) with corresponding openings 20 in the substrate 2. The cover 14 has a first reading window 22 and a second reading window 24, through which the contents of the chambers of the substrate 2 below can be read and thereby analyzed (e.g., by fluorescence detection) or at least inspected.

[0032] In one optional variant (shown here), the disc 1 also comprises a (here two-part, preferably adhesive) label 26 attached to the cover 14. The label 26 is configured to be readable through the reading windows 22 and 24. In a further optional form of this variant, the label 26 has transparent areas covering the reading windows 22 and 24. Preferably, such transparent areas are not provided with adhesive, i.e. no adhesive adheres to them, so that the fluorescence can be detected without the influence of the luminescent adhesive.

[0033] The cover 14 has recesses 28 formed in the sides of the side walls 30 to allow the disk 1 to be aligned and positioned within the automatic feeder of the analyzer.

[0034] The substrate 2 has a plurality of (here specifically, two) perforations 32 which are used to clearly align and position the disk 1 on a support plate of an analytical device (hereinafter referred to as "turntable 34", see Figures 3 to 5). Positioning pins 38 of the turntable 34 engage with these perforations 32 to position and fix the disk 1 on a rotation surface 36 which is parallel to the upper surface of the turntable 34 and the heat input side 8 of the disk 1 (hence the planar extension).

[0035] The turntable 34 of the analytical device is used for centrifugation, i.e., for rotating the disk 1 around a rotation axis 40 (see FIG. 4). The turntable 34 is optionally configured to accommodate two disks 1 and is therefore formed with 180-degree rotational symmetry (see FIG. 3). Furthermore, the turntable 34 supports a number of heating elements 42, which are used for localized heating of the individual chambers of the channel and chamber structure 4 of the disk 1 and therefore have their outer contours adapted to the corresponding chambers. In this embodiment, the heating elements 42 are formed by resistive heating plates.

[0036] In an alternative embodiment, the heating element 42 is formed by a Peltier element, which also allows for active cooling.

[0037] The individual chambers, channels and further elements of the disk 1 will be explained in more detail with reference to the method sequences described below.

[0038] To perform the analysis method, at least one disk 1 is introduced into the analysis device and positioned on the turntable 34. A swab 44 is inserted into the disk as a sample material carrier through the access 10 into the swab chamber 46 of the channel-and-chamber structure 4, which is then closed by the capsule 12. When only one disk 1 is installed, the analysis device is configured to automatically balance the turntable 34 (in particular by providing a counterweight on the turntable 34). For fixation, the disk 1 is attracted to the turntable 34 by a vacuum pump. For this purpose, the heating element 42 is provided with a sealing contour 48 around its periphery. The disk 1 is in contact with this contour, which allows it to be attracted to the turntable 34 in a specific area. This advantageously allows for close contact between the heating element 42 and the locally heated portion of the disk 1.

[0039] In its initial state (i.e., without a sample or swab 44 already inserted), the disc 1 contains pre-stored additives or analytes in the form of liquid reagents in the sealed stick packs 50 in the first stick pack chamber 52 and the second stick pack chamber 54. Furthermore, so-called primers are also pre-stored as additives or analytes in the pre-amplification chamber 56. Furthermore, primers and so-called probes (also known as "gene probes", usually in the form of polynucleotides or oligonucleotides) are pre-stored in a number of reading chambers 58. These reading chambers 58 are visible through the reading window 24 in the cover 14. The primer pairs in the pre-amplification chambers 56 may be identical or different depending on the specific purpose and / or specific steps of the analytical method. For example, the primers in the reading chambers 58 may be pairwise identical to the primers in the pre-amplification chambers 56, or may be different and therefore provided for, for example, "nested PCR" ("nested" or "embedded" PCR), as known in the art.

[0040] The first, roughly circular "lyophilization chamber 60" and the second, roughly circular "lyophilization chamber 61" contain pre-stored lyophilized materials, including, for example, enzymes, polymerases, dNTPs (deoxynucleotide triphosphates), salts, and / or additional pre-stored reagents (e.g., PCR additives, nuclease inhibitors, cofactors for participating enzymes, etc.). In this embodiment, the swab chamber 46 contains a lysis agent and a process control agent, such as spores, fungi, phages, or artificial targets. The lysis chamber 62, which is connected to the swab chamber 46, contains a lysis pellet as well as a magnet and grinding media, such as glass and / or zirconia particles. These particles are optionally coated with EDTA, or EDTA is added to prevent coagulation when blood is used as the sample material. Activated carbon is optionally added to bind inhibitors; in this case, activated carbon is also pre-stored.

[0041] After sample collection, a sample carrier, in this case a swab 44 (also called a "swab" in the medical field under sterile conditions), is inserted into the disk 1, specifically the swab chamber 46, using, for example, a blood capillary, and the access 10 is closed by the capsule 12. The capsule 12 is then closed airtight to prevent the escape of pathogens possibly contained in the sample material. In an optional embodiment, the disk 1, specifically the base 2, has a vent hole 64, upstream of which is arranged a filter and condensation trap 66 (in the form of a relatively small chamber). The latter allows the condensation water to wet the filter. However, the vent hole 64 can be omitted in an alternative embodiment.

[0042] After the disk 1 is positioned and secured on the turntable 34, lysis of the sample material is initiated by moving a magnet located in the analyzer above the disk 1. This generates a magnetic field that changes relative to the frame of reference of the disk 1, causing the magnet located in the lysis chamber 62 to move. The movement of the magnet causes particles of grinding media present in the lysis chamber 62 to rub against each other, thereby solubilizing bacteria, fungi, viruses, or other analytes.

[0043] This mechanical lysis is, in an optional method step, thermally supported by heating the lysis chamber 62 by means of corresponding locally assigned heating elements 42 .

[0044] Meanwhile, the turntable 34 rotates, which also rotates the disc 1, and larger sample particles are sedimented by centrifugation, thereby increasing the resistance to biochemical inhibition and reducing the risk of clogging the microfluidic channels of the channel and chamber structure 4.

[0045] Optionally, the sample can already be replicated in this first step by polymerase chain reaction (PCR) or by isothermal methods (e.g. loop-mediated isothermal amplification, abbreviated: LAMP, or recombinase polymerase amplification, abbreviated: RPA). Non-specific amplification in this first step is also conceivable, for example by so-called whole genome amplification based on PCR or MDA (multiple displacement amplification).

[0046] Typically, however, the sample material is first homogenized by the magnet and particle movement, optionally supported by convection based on a temperature gradient generated in the lysis chamber 62 by optional one-sided heating. If a biochemical reaction is additionally performed in the lysis chamber 62, the reaction conditions in the lysis chamber 62 are simultaneously maintained homogenous, i.e., a particularly stable temperature distribution is established and / or high material mixing is achieved. This is particularly suitable for samples with very low concentrations or samples that are difficult to lyse. Possible shearing of DNA or RNA also reduces secondary structures, thereby supporting subsequent amplification. The mechanical action of the moving magnet and the force applied to the sample can (randomly) cleave ("shear") the DNA or RNA strands. The degree of shearing can be controlled by the duration and intensity of the mechanical action (i.e., "mechanical lysis"), e.g., the speed of the magnet movement. However, care must be taken, as excessive shearing of DNA and RNA can prevent amplification.

[0047] In a further method step, the stick pack chambers 52 and 54 are locally heated to about 90°C by the corresponding heating elements 42, and then (optionally during this time) the rotation speed of the turntable is increased to above 30Hz, in particular to about 60Hz. During centrifugation at this medium to high rotation speed, the combination of heating and centrifugal force causes the stick pack 50 to open in a relatively short time, such as around 5 seconds. This heating thermally weakens the tear or peel seam of the stick pack 50, which is made from a so-called peel film.

[0048] During rotation at a frequency of at least 25 Hz, in particular at least 30 Hz as mentioned above, and preferably at about 60 Hz, an overpressure builds up in the Stick-Pack chambers 52 and 54 due to heating. This overpressure is caused not only by the expansion of the gas contained in each Stick-Pack chamber 52 or 54 (due to the ideal gas law), but also by the vapor pressure, which depends on the Stick-Pack liquid and the temperature in the Stick-Pack chamber 52 or 54. Subsequently, upon slowing down of the rotation speed, this overpressure causes a large portion of the liquid (preferably more than 90%) to be expelled from the Stick-Pack chamber 52 or 54 through the connecting channel 68 into the lysis chamber 62 or the lyophilization chamber 61, respectively. This expulsion is robust against the centrifugal forces within the disk 1, even during centrifugation at frequencies between 10 and 30 Hz. The liquid expelled from the Stick-Pack chamber 54 dissolves the lyophilizate placed in the lyophilization chamber 61, which contains part of the reagents for the subsequent primary amplification. This liquid transfer to the lysis chamber 62 or freeze-drying chamber 61 takes place before or during the (mechanical) lysis described above in the lysis chamber 62 in order to make the liquid in the stick pack 50 in the stick pack chamber 52 available for use.

[0049] Figure 8 shows an opened stick pack 50 and the hatched area shows liquid that has leaked from the stick pack 50 into the stick pack chamber 54. Some of the liquid has already escaped from the stick pack chamber 52 into the swab chamber 46 and dissolution chamber 62. Figure 9 shows the state of the stick pack chambers 52 and 54 after the liquid has been expelled from each.

[0050] In a subsequent method step, the liquid ("lysate") is transported from the lysis chamber 62 to the subsequent freeze-drying chamber 60 (shown above and to the right of the lysis chamber 62 in FIGS. 8-11), where it dissolves the lyophilized material previously stored therein (see FIG. 10). This lyophilized material may optionally contain, in addition to the amplification reagents described above, nuclease inhibitors for inactivating specific nucleases, as well as additives or cofactors such as dithiothreitol (DTT). The transport of the lysate is then achieved by heating the upstream Stick Pack chamber 52 and / or the lysis chamber 62, and / or by cooling the pre-amplification chamber 56, the (opposite) Stick Pack chamber 54, and / or the read chamber 58, which are fluidly connected downstream of the freeze-drying chamber 60, against the centrifugal force caused by further rotation of the disk 1. Cooling the subsequent chambers creates a suction effect due to negative pressure, while heating the preceding chamber or chambers correspondingly and conversely creates a liquid advance due to overpressure.

[0051] The freeze-drying chamber 60 is connected to an overflow chamber 72 by an overflow channel 70. During the transfer of lysate from the lysing chamber 62 to the chamber 60, excess lysate flows through the overflow channel 70 into the overflow chamber 72. Control chambers 74 and 76 are connected to the overflow chamber 72 and are used to verify whether the disk 1 is properly filled. The lysate flowing into the overflow chamber 72 is then filled into the control chamber 74 and then into the control chamber 76 (schematically shown in FIG. 10 ). The filling of the control chambers 74 and 76 is used to verify the proper filling of the disk 1. In particular, the filling of the control chamber 74, which has a substantially square shape and is shown on the right side of FIG. 10 , and specifically the control chamber 76 connected radially outward thereto, is interpreted as meaning that the disk 1 is not underfilled. On the other hand, filling the roughly triangular control chamber 74 (see FIG. 10 ) fluidly adjacent to the roughly square control chamber 74 on the left, specifically the control chamber 76 connected radially outward of the control chamber 74, is interpreted as overfilling the disk 1. The total amount of liquid present therein is composed of the volume of the introduced sample material, typically about 105-170 microliters, and the volume of the Stick Pack 50 in the Stick Pack chamber 52, typically about 140-160 microliters. The filling of the corresponding control chambers 74 and 76 can be continuously monitored by a fluorescence detector through the reading window 22 in the cover 14 at specific times (e.g., at the end of the entire analytical method) or even during the filling of the lyophilization chamber 60. This allows the time when the control chambers 74 and 76, and thus the lyophilization chamber 60, are filled to be known. This, in turn, allows for the estimation of possible sources of error. In one optional embodiment, a dried fluorescent dye is incorporated into the control chambers 74 and / or 76 to obtain a stronger signal.

[0052] In a further method step, the liquid is then transported from the freeze-drying chamber 60 through the transfer channel 78 to the pre-amplification chamber 56 at a high rotation speed of 40-60 Hz. As soon as the liquid state in the pre-amplification chamber 56 exceeds the opening of the respective outlet channel 80, the trapped air volume is compressed in the (radially inward) "headspace" of the pre-amplification chamber 56 and in the respective chamber 84 located downstream via the assigned channel 82 (see FIG. 11 ).

[0053] In the next method step, preamplification is performed in the preamplification chamber 56 under high-speed centrifugation at a rotation speed of 40-80 Hz. The overpressure in the preamplification chamber 56 and chamber 84 is maintained by high-speed centrifugation during preamplification. First, previously stored primers, e.g., spotted with trehalose, are decomposed in the preamplification chamber 56. For RNA detection, optional reverse transcription can be performed first at a constant temperature of 35-70°C for 30 seconds to 10 or 30 minutes to transcribe the RNA present into DNA. However, PCR preamplification is performed by localized and cyclical heating and cooling of the liquid in the preamplification chamber 56 within the ranges of 50-75°C and 80-100°C. Preamplification involves 5-30 preamplification cycles. Each cycle involves heating to 80-100°C and then cooling to 50-75°C.

[0054] The (pre-amplification) reaction in the pre-amplification chamber 56 is supported by significant convection. This is due to the unilateral heat input from the disk 1, specifically the heat input side 8, to the pre-amplification chamber 56 and the simultaneous rotation. As can be seen from FIGS. 12 and 13, the liquid in the pre-amplification chamber 56 is first heated at the heat input side 8, which is heated by the heating element 42, thereby forming a heated boundary layer. The density of the boundary layer is then lower than that of the rest of the liquid volume. The heated liquid in the boundary layer rises in the artificial gravitational field oriented in the radial direction R by the rotation of the disk 1, first "inward" with respect to the radial direction R and then toward the upper side 16 transverse to the radial direction R. There, the liquid cools and, "by gravity," moves outward along the upper side 16 in the radial direction R and sinks back to the heat input side 8 (see FIG. 13). Thus, the heat input generates convection and flow along the radial direction R. 12 (plan view from the upper side 16) and 13, the temperature and density distributions are shown schematically by different hatchings. Furthermore, the Coriolis force also generates a tangential flow component (i.e., perpendicular to the radial direction R in the plane of the disk 1), which further supports the mixing of the liquid. Since the convection is induced by the artificial gravitational field, the faster the disk 1 rotates, the stronger the convection. Therefore, the convection generated at high rotation speeds leads to particularly effective mixing of the reaction components in the pre-amplification chamber 56, which in turn leads to efficient amplification conditions.

[0055] However, as a side effect, if there is very high heat release on the unheated top side 16 of the disk 1, a large temperature gradient of, for example, 10°C (or Kelvin) can form within the liquid volume of the pre-amplification chamber 56, which can be detrimental. In the embodiment shown in Figure 13 (without the cover 14), when the heating element 42 is controlled to heat at 97°C, the temperature value in the lower left region of the pre-amplification chamber 56 (hatched diagonally upward to the right) is 95°C, and the temperature value in the upper right region of the pre-amplification chamber 56 is 85°C.

[0056] Such a large temperature gradient is reduced in the embodiment shown in Figure 14 by the cover 14 which provides an air shield, so that with the same heat input, the temperature values ​​are 95°C in the lower left area and 91°C in the upper right area, thereby achieving a difference of 4 Kelvin.

[0057] To further reduce heat emission, in a further embodiment, the cover 14 has a frame web 86 that surrounds the pre-amplification chambers 56 in a ring-like manner, thereby further reducing heat emission due to convection at the upper side 16 (see FIGS. 1, 7, and 15). The frame web 86 is molded to the cover 14, i.e., is integrally connected to the cover. The frame web 86 protrudes toward the base 2 and terminates at a small distance of approximately 100 μm from the base. The frame web 86 also surrounds the plateau-shaped pre-amplification chambers 56 at their sides at the upper side 16. This continuous shielding of the pre-amplification chambers 56 by the cover 14 and the frame web 86 allows a temperature difference of approximately 2 Kelvin within each pre-amplification chamber 56, i.e., between the two different hatched areas in FIG. 15. This allows for relatively strong convection, particularly due to rotation, to occur in each pre-amplification chamber 56. In addition, however, at least in the static case, i.e., when the temperature of the heating element 42 is maintained for at least about 10-30 seconds, a relatively high homogeneity of the reaction temperature is achieved in the pre-amplification chamber 56. Experience has shown that with the geometry and parameters used in this embodiment and those described below, static conditions can be achieved already after about 15 seconds.

[0058] When reactions requiring interactions are performed in the pre-amplification chamber 56, such as binding of molecules to a solid phase, e.g., binding to a microarray, or reactions in which the concentrations of the respective reaction partners are typically low and therefore the probability of contact between the respective reaction partners is relatively low, a large convection (and therefore relatively strong mixing) as well as a uniform temperature distribution can be advantageous.

[0059] After the pre-amplification is completed, the rotation speed is reduced to approximately 5-20 Hz, specifically approximately 10 Hz, thereby expanding the amount of compressed air in the headspace of the pre-amplification chamber 56 and in the chamber 84. This, in turn, results in a decrease in the liquid level in the pre-amplification chamber 56, since the liquid radially present in the opening of the discharge channel 80 is expelled, at least in large part, into another chamber 88 by the expanding air passing through the discharge channel 80. This is possible because the discharge channel 80 has a lower flow resistance, specifically a larger channel cross-section, than the transfer channel 78 leading to the pre-amplification chamber 56.

[0060] In this regard, the disk 1 is particularly provided with a vent channel 90 that communicates with the chamber 88 and allows internal venting to the other chambers of the disk 1. Thereby, air compressed by the liquid flowing into the chamber 88 can escape via the vent channel 90 in the direction of the freeze-drying chamber 60.

[0061] In the next method step, the rotation speed of the disk 1 (or turntable 34) is set to a value in the range of 10 to 20 Hz, preferably about 15 Hz, and specifically increased. The liquid from the chamber 88 flows via a siphon 92 into a measuring chamber 94, which has three radially outwardly disposed "measuring fingers" or chamber projections of different volumes. These fingers are then filled one after the other, thereby measuring individual partial volumes based on a given (measuring finger) volume (see FIG. 16). The flow of liquid through the channels 96 radially outward of the measuring fingers is limited by their high flow resistance, specifically by their channel dimensions in at least one spatial direction, specifically the cross-sectional direction, being less than 200 μm. Therefore, the partial volumes flowing through the channels 96 in the next step are essentially predetermined by the volume of the respective upstream measuring finger. The excess liquid flows to a drain 98.

[0062] In the next method step (see Figure 17), by increasing the centrifugation, i.e. increasing the rotation speed to the range of 30-80 Hz, specifically to about 60 Hz, the measured partial volume of liquid is transferred to the respective subsequent chamber, i.e. to the freeze-drying chamber 61 shown to the left of the swab chamber 46 in Figure 17, and then to chamber 100.

[0063] This freeze-drying chamber 61 contains a "main amplification buffer" that was originally stored in advance in a stick pack 50 placed in the stick pack chamber 54, a freeze-dried material that was previously stored in the freeze-drying chamber 61 and dissolved in liquid during that time, and a "pre-amplification product" contained in the liquid from the pre-amplification chamber 56 that is supplied via channel 96.

[0064] In a subsequent method step, the disk 1 is rotated with relatively rapid direction changes between end values ​​of -20 to 40 Hz and +20 to 40 Hz, with a change rate of 5 to 40 Hz / s, preferably about 30 Hz / s, respectively. The signs in this case indicate the difference in the direction of rotation. The components present in the freeze-drying chamber 61 are mixed by the accelerations that occur during the direction changes and the Euler and Coriolis forces that are generated in the rotating system.

[0065] In parallel, the reading chambers 58, the upstream measuring chambers 102, and the overflow chamber 104 are heated by the corresponding heating elements 42. This allows the expanded air to escape into the chamber 100 via the compensation channel 106. At the end of the mixing process, the direction change is terminated and the rotation speed is again set to a constant speed of approximately 20 Hz. The reading chambers 58, measuring chambers 102, and overflow chamber 104 are then cooled again. This creates a relative negative pressure in each chamber 58, 102, and 104, increasing the filling state of the siphon channel 108 downstream of the freeze-drying chamber 61 and the compensation channel 106 connected to the chamber 100, depending on the ratio of the centrifugal pressure to the air pressure difference. As soon as the liquid exceeds the apex 110 of the siphon channel 108, all of the liquid moves from the freeze-drying chamber 61 to the downstream measuring chamber 102 (see FIG. 18). A vent channel 112 between the freeze-drying chamber 61 and the chamber 100 then allows air exchange between these two chambers 61 and 100 .

[0066] In doing so, the liquid flows sequentially into the individual measuring chambers 102 and is thereby measured. Furthermore, the liquid in each measuring chamber 102 is held back by a centrifugal pneumatic valve in the form of a valve channel 114. Excess liquid flows into the overflow chamber 104. The centrifugal pneumatic valve is based on the following: the liquid is held in the respective valve channel 114 by the counter pressure in the respective subsequent reading chamber 58 and cannot flow into the subsequent reading chamber 58 at medium rotational speeds, in particular at rotational speeds of about 15-25 Hz in this case.

[0067] In a subsequent method step, the rotation speed is increased, typically to above 40 Hz, until the meniscus in each valve channel 114 becomes unstable due to the so-called "Rayleigh-Taylor instability", thereby transferring at least a large portion of the liquid to the corresponding reading chamber 58 (see Figure 19).

[0068] In a further method step, the main amplification is carried out in the reading chambers 58. For this purpose, the primers and probes previously stored in the reading chambers 58 are dissolved. The dissolution of the primers and probes and the subsequent amplification are supported by high convection in the reading chambers 58, as described above with reference to Figures 12 and 13. The reaction is read out in all reading chambers 58 after each cycle at approximately 60°C by a fluorescence detector. The fluorescence detector detects fluorescence of different wavelengths. Reading is performed through the reading window 24 in the cover 14. This process corresponds to so-called "real-time PCR." Multiplex reactions, for example, 3- to 10-plex, can be carried out in each of the 12 reading chambers 58. A corresponding increase in the fluorescence detector signal indicates the detection of a target.

[0069] In order to avoid or minimize the influence on the optical evaluation by the fluorescence detector, the reading chamber 58 has a recess (not shown in more detail) radially inward, outside the observation area of ​​the fluorescence detector, which serves to "trap" the gas bubble and prevent it from entering the observation area. A gas bubble in this recess must be relatively strongly deformed in order to avoid entering the observation area. Advantageously, in this embodiment, the boundary surface of the gas bubble is influenced in particular by the existing surface tension conditions, which exert a counteracting effect.

[0070] Furthermore, as an option, in this case the transparently closed reading window 24 covering the reading chamber 58 is also bordered by a frame web equivalent to the frame web 86 (see Figure 1), thereby also reducing heat emissions from the reading chamber 58.

[0071] Instead of fluorescence detection, evaluation can also be performed using so-called melting curve analysis, such as "high-resolution melting curve analysis" or "rapid melting curve analysis." This allows for even higher multiplexing. In one optional embodiment, "real-time PCR" based on so-called intercalating dyes (e.g., dyes known under the brands or names "EvaGreen," "SYBR Green," or "BoxTo") is performed in the reading chamber 58, whereby the resulting PCR products are detected by melting curve analysis after amplification. Up to 20 PCR products can be detected and distinguished per chamber (20-plex).

[0072] 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]

[0073] 1 disc 2 Base 4. Channel and chamber structure 6. Sealing film 8 Heat input side 10 Access section 12 capsules 14 Cover 16 Upper side 18 Locking hook 20 Opening 22 Reading window 24 Reading window 26 Label 28 Recess 30 side wall 32 perforation 34 Turntable 36 Surface of Revolution 38 Locating pin 40 Rotational Axis 42 Heating element 44 swabs 46 Swab Chamber 48 Seal contour 50 stick packs 52 Stick Pack Chamber 54 Stick Pack Chamber 56 Pre-amplification chamber 58 Reading Chamber 60 freeze-drying chambers 61 Freeze-drying chamber 62 Melting Chamber 64 vent holes 66 Condensate trap 68 channels 70 Overflow Channel 72 Overflow Chamber 74 Control Chamber 76 Control Chamber 78 Transfer Channel 80 Discharge Channel 82 channels 84 Chamber 86 Frame Web 88 Chamber 90 Vent Channel 92 Siphon 94 Measurement Chamber 96 channels 98 Drain 100 Chambers 102 Measurement chamber 104 Overflow Chamber 106 Compensation Channels 108 Siphon Channel 110 Vertex 112 Vent Channel 114 Valve Channel R Radial direction

Claims

1. A cartridge (1) for a rotation-based analytical method using one-sided heat input, comprising: a planar substrate (2) having a microfluidic channel and chamber structure (4) formed therein, the processing chambers being interconnected by the channels; a plurality of positioning and / or fixing elements (32) formed on the substrate (2) for positioning and / or fixing the substrate (2) to a support plate (34) of an analytical device for carrying out the analytical method; a cover body (14) fixed to the base body (2), the cover body (14) being disposed on one side of the upper side (16) of the base body (2) opposite the heat input side (8) and covering at least one chamber (56); The substrate (2) comprises a base material having a channel and chamber structure (4) inserted therein, and a seal coating (6) sealing the channel and chamber structure (4); The cartridge (1), wherein the cover body (14) has a frame web (86) that projects toward the upper side (16) of the base (2) and surrounds the covered chamber (56).

2. 2. The cartridge (1) according to claim 1, wherein the chamber covered by the cover body (14) is an amplification chamber (56) for the replication of genetic material.

3. 3. A cartridge (1) according to claim 1 or 2, wherein the cover body (14) at least almost completely covers the upper side (16) of the base body (2).

4. A cartridge (1) according to any one of claims 1 to 3, wherein the frame web (86) rests on the base body (2) in the intended state of use or is arranged at a small distance from the base body (2), in particular at a distance of less than 0.5 mm, preferably 0.1 mm or less.

5. The cartridge (1) according to any one of claims 1 to 4, wherein the covered chamber (56) protrudes upward on the upper side (16) of the base (2) and partially overlaps the frame web (86).

6. A cartridge (1) according to any one of claims 1 to 5, wherein the cover body (14) is made from a plastic, in particular a thermoplastic.

7. A cartridge (1) according to any one of the preceding claims, wherein the base (2) comprises a thermoplastic substrate.

8. A rotation-based analysis method comprising: A cartridge (1) according to any one of claims 1 to 7 is fixed to a support plate (34) of an analytical device, The support plate (34) is rotated while holding the cartridge (1), According to each method step, heat is introduced into the substrate (2) on one side by a predetermined number of heating elements (42) arranged on the support plate (34), An analysis method, wherein the rotation speed is varied between a low rotation speed range up to 20 Hz, a medium rotation speed range from 20 to 40 Hz, and a high rotation speed range from 40 Hz, depending on the respective method steps.

9. A polymerase chain reaction is carried out in the chamber (56) covered by the cover body (14); 9. The analytical method according to claim 8, wherein the temperature of the liquid in the chamber (56) is set by cyclic heat input at 50-75°C on the one hand and at 80-100°C on the other hand, and medium to high rotation speeds are used.

10. A method of using a cartridge (1), comprising: Use of a cartridge (1) according to any one of claims 1 to 7 in a rotation-based analytical method according to claim 8 or 9.

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