Method for operating an analyzer, method for using a cartridge, and analyzer

Abstract

In a method for operating an analytical device, particularly for performing analytical processing by polymerase chain reaction, a cartridge (1) having a microfluidic channel and chamber structure (4) is provided. At least one film pouch (50) containing a processing liquid is placed in a stick pack chamber (52, 54) of the cartridge (1). In an opening step, the at least one stick pack chamber (52, 54) is heated to a temperature of 80 to 130°C, and in the opening step, the cartridge (1) is rotated at a rotation speed of 20 to 80 Hz.

Description

[Technical field]

[0001] The present invention relates to a method for operating an analytical device and in particular to the use of a cartridge in such a method. Furthermore, the present invention relates to such an analytical device. [Background technology]

[0002] Analysis methods based on rotation are used in the medical field, in particular under the use of so-called cartridges with microfluidic channel and chamber structures. They are mainly used for the scientific analysis of genetic material, mainly in the form of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), for the analysis of said genetic material for the testing of existing diseases or generally for the detection of pathogens. For this purpose, it is necessary to replicate specific parts of the genetic material (DNA or RNA) contained therein from a sample, for example a mucosal tissue or blood sample. When RNA in a sample is detected or analyzed (for example for the detection of viruses), this 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 usually 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, usually to between 90 and 96 °C.

[0004] The temperature is then lowered again ("annealing step", usually in the range of 50-70°C) to allow specific attachment of so-called primer molecules to the single strands. Primer molecules are short complementary DNA strands that bind to the single stranded DNA at defined positions. The primer molecules (also "primers" for short) serve as starting points for the enzymes called polymerases, which fill in the existing DNA sequence of the single strand with complementary building blocks (dNTPs) in the so-called extension step. In this process, a double stranded DNA is formed again, starting from the primer molecules. The 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] Such cycling of the temperature in the liquid reaction mixture through two to three temperature ranges is called PCR thermal cycling, and is usually repeated 30 and 50 cycles. In each cycle, a specific DNA region is replicated. Generally, the thermal cycle of the liquid reaction mixture is reversed by controlling the external temperature in the reaction vessel. The reaction vessel is, for example, placed in a thermoblock, in which the PCR thermal cycle is reversed by heating and cooling a solid in thermal contact with the reaction vessel, whereby heat is supplied to and removed from the liquid. Alternatively, heating and cooling concepts for the reverse of the PCR thermal cycle are temperature control of the fluid (particularly air and water) flowing around the reaction vessel, as well as radiation-based concepts by heat supply, for example, using ultraviolet or laser irradiation. In the case of rotation-based methods, a chamber in the cartridge, for example as described above, is used as the reaction vessel, which is correspondingly heated. Furthermore, a cartridge formed in an approximately disk shape is usually rotated. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to further improve the analytical method. [Means for solving the problem]

[0007] This problem is solved according to the invention by a method for operating an analytical device having the features of claim 1. Furthermore, the problem is solved by the use of a cartridge having the features of claim 7. Furthermore, the problem is solved according to the invention by an analytical device having the features of claim 9. Advantageous, partly inventive per se, embodiments and further configurations of the invention are set out in the dependent claims and the following description.

[0008] The method according to the invention is used for operating an analytical device, which is configured and intended in particular for carrying out an analytical process (or: analytical method) by polymerase chain reaction. According to the method, a cartridge, which in particular represents a sample carrier and has a microfluidic channel and chamber structure, is prepared. At least one film pouch (hereinafter also referred to as "stick pack") containing a processing liquid is then placed in a so-called (and preferably corresponding to one of a plurality of stick packs) stick pack chamber (in particular channel and chamber structure) of the cartridge. In an opening step (of the operating method described herein), at least one stick pack chamber (in particular each containing one stick pack) is heated to a temperature of 80 to 130 ° C. Furthermore, in this opening step, the cartridge is rotated at a speed of 20 to 80 Hz.

[0009] Thus, at least one stick pack is preferably used to hold the processing liquid in the cartridge for the analytical process. The stick pack then has the advantage that it is relatively easy to handle when mounting the cartridge. The cartridge is formed by a substrate in which in particular a channel and chamber structure is formed. This substrate is at least partially covered, i.e. closed, in particular sealed by a sealing film (preferably with the exception of an access opening for introducing the sample material and, if present, a vent opening) after mounting by the or each stick pack and possibly other, optionally dry, processing media. Processing media include, for example, dried fluorescent dyes or so-called primers which react with certain substances and thereby allow the evaluation of the analytical process by fluorescence detection. Alternatively, such processing media are also referred to as "analytes". Dry processing media are relatively easy to handle, since they are usually not flowable. Furthermore, the above-mentioned treatment method in the opening step, i.e. the combination of a relatively high temperature and a relatively high rotation speed, advantageously allows a rapid opening of the or each stick pack, which on the other hand also shortens the processing time.

[0010] The term "microfluidic" or "microfluidic channel and chamber structures" is understood here and below to mean in particular that the components, preferably at least the channels, have at least one dimension, in particular the depth or width, in the range of 30 to 700 micrometers. In the case of channels, preferably in both depth and width directions, they are of this order of magnitude. Some chambers may then have larger dimensions.

[0011] Preferably, in the opening step, a temperature of about 60-120°C, for example about 95°C, is selected with a rotation speed of at least 30Hz, preferably about 60Hz, which allows the stick pack to be opened within about 5 seconds.

[0012] In a preferred variant of the method, a bag made of so-called peel film is used as the stick pack. This bag, on the other hand, also has a preferably heat-sealed separation seam (or also called "peel seam"). In other words, the stick pack is thermally sealed. A thermally supported opening is therefore advantageous. "Peel film" is understood here and below to mean a film that is designed and formed to form a relatively easily detachable bond with itself and / or with other materials. The seam strength of the separation seam is therefore advantageously lower in any case than the welded seam strength of conventional films. As material for the peel film, in particular polypropylene or polyethylene is used.

[0013] In a further preferred variant of the method, the or each stick pack chamber is heated locally, which has the advantage that the sample material or additional processing medium is not affected by heating during the opening step, which further allows the energy requirements of the method to be kept as low as possible.

[0014] In one preferred variant of the method, the volume of the stick pack chamber and the liquid amount of the respective film pouch are selected such that said liquid amount in an undrained state is (or occupies) less than or equal to about ¼ of the volume of the stick pack chamber. "Undrained state" is understood here and below to mean in particular that the liquid amount of the stick pack is located in the stick pack chamber and has not yet been drained from the stick pack chamber through the channel.

[0015] In a further preferred embodiment of the above method variant, a volume of about 1000-1500 microliters is contemplated for the stick pack chamber, and therefore a liquid volume of about 150-375, preferably up to about 250 microliters is contemplated for the stick pack.

[0016] Preferably, the stick pack chamber is arranged radially outward with respect to the axis of rotation of the cartridge, i.e. the axis of rotation about which the cartridge is rotated (which does not necessarily lie in the area covered by the cartridge). Preferably, the (or each) stick pack chamber is then arranged offset with respect to the outer edge of the cartridge. This allows the relatively voluminous stick pack chamber to be arranged as space-efficiently as possible, especially in the case of a cartridge shape which corresponds approximately to a semicircular plate. In particular, as the cartridge rotates during the execution of the analysis method, the liquid coming out of the stick pack also gathers at the outer edge of the stick pack chamber due to centrifugal forces. The or each stick pack chamber is therefore connected by a channel connected radially outward to the stick pack chamber to a "following" chamber arranged offset radially inward. In order to empty the stick pack chamber and transfer the liquid to this following chamber through the channel connected radially outward, now in a preferred variant of the method, the rotation speed is reduced, especially at a temperature which remains constant, preferably in relation to the opening step of the stick pack. The temperature increase for opening the stick pack causes an internal pressure to increase in the stick pack chamber, as the gas in the stick pack chamber, e.g. air or inert gas, expands due to the temperature. The internal pressure is further increased by the sublimating liquid, due to its vapor pressure, which depends on the type of liquid (in the stick pack) and on the temperature. However, since the subsequent chamber is offset radially inwards, in the channel leading to this chamber, the centrifugal force acts against the liquid "displaced" by the internal pressure of the stick pack chamber. However, when the rotation speed is reduced, the centrifugal force and therefore the "gravitational pressure" or "centrifugal pressure" that pushes the liquid radially outwards in the channel is reduced, so that the liquid displaced by the internal pressure of the stick pack chamber can also flow in the channel against the centrifugal force to the subsequent chamber. If the internal pressure is high enough, the stick pack chamber can be at least almost completely emptied, e.g. except for condensation.Preferably, the channel is then connected radially inwards to the subsequent chamber, whereby the liquid transported through the channel can advantageously flow out of the channel outlet, in particular also because this subsequent chamber has a larger chamber volume compared to the liquid amount in the stick pack. This reduces the risk of the liquid remaining in the subsequent chamber being "splashed up" by the air flowing in from the stick pack chamber. The internal pressure of the stick pack chamber is then optionally controlled in a time-dependent manner, i.e. by maintaining the temperature for a relatively long time at high rotation speeds or by additionally heating the stick pack chamber before lowering the rotation speed. In contrast, during the transport of the liquid, the temperature must be kept constant in order to avoid cooling of the stick pack chamber and thus changes in the pressure relationship.

[0017] Preferably, the rotation speed is reduced to about 5-20 Hz, in particular about 10-15 Hz. This rotation value increases the probability that the stick-pack chamber can be completely (at least almost) emptied as described above. However, in particular, the rotation speed and the internal pressure of the stick-pack chamber influence each other, depending in particular on the radial position of the channel inlet relative to the radial position of the channel outlet in the subsequent chamber (i.e. at which radial position the channel is connected to the stick-pack chamber). The greater the radial distance between the channel inlet and the channel outlet, the higher the (internal) pressure is required to transport the liquid through the channel. However, the rotation speed cannot be reduced arbitrarily, because otherwise the liquid in the stick-pack chamber would not be "pushed" reliably enough towards the channel inlet.

[0018] By having the subsequent chamber disposed radially inwardly of the stick pack chamber, or at least initially connected to it by a channel running radially inwardly in a curved manner, advantageously liquid previously stored in the stick pack initially remains in the stick pack chamber after it has been opened, so that it can be precisely transferred to the subsequent chamber by the measures described above (possibly also at a later time).

[0019] The invention also relates to the use of a cartridge, as described here and in more detail below, in the above-mentioned method. The cartridge comprises a microfluidic channel and chamber structure as described above, in particular at least covered and preferably liquid-tight sealed except for an access for introducing a sample substance and an optional vent opening. This channel and chamber structure comprises at least one stick pack chamber. The cartridge further comprises at least one stick pack containing a processing liquid as described above and arranged and in particular fixed in the stick pack chamber. Preferably, the or each stick pack is then glued or otherwise fixed to the corresponding stick pack chamber. Furthermore, the cartridge comprises a flat disc-like geometry. Preferably, the structure approximates a half-disk. As described above, the or each stick pack chamber is further arranged radially outward with respect to the axis of rotation about which the cartridge rotates during the execution of the method. The or each stick pack chamber is connected by a radially outwardly connected channel to a radially inwardly offset arranged ("subsequent") chamber. Alternatively, this subsequent chamber may also be located at the same radial position as the radially outer edge of the Stick Pack chamber or further outwards than the radially outer edge, in which case the channel connecting these two chambers curves radially inwards starting from the channel inlet of the Stick Pack chamber as described above, with this channel being dimensioned (in particular "curved" as far as possible radially inwards) in order to prevent the liquid column standing in this channel from prematurely or uncontrolled emptying of the Stick Pack chamber into the subsequent chamber due to the centrifugal forces as described above.

[0020] The analytical device according to the invention is configured and intended for carrying out an analytical procedure, in particular based on rotation, in particular by polymerase chain reaction. To that end, the analytical device comprises a receiving device for the cartridges as described above. Preferably, this receiving device comprises an automatic feeder, in particular similar to a CD drive. Preferably, the receiving device also comprises a support plate on which the cartridges are fixedly placed. This support plate is preferably formed as a turntable. In order to rotate the cartridges (in particular, preferably the turntable), the analytical device comprises a rotation drive. Furthermore, the analytical device comprises a heating device for a particularly locally limited heat input to the cartridges. Preferably, this heating device is formed by a number of heating elements, for example resistive heating plates or Peltier elements, arranged locally on the support plate. Furthermore, the analytical device comprises a control device, which is configured to carry out the above-mentioned method for operating the analytical device, in particular automatically.

[0021] Preferably, the control device is then 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 implemented programmatically in the form of operating software (firmware), so that when the operating software is executed in the microcontroller, the method is executed automatically, if necessary in dialogue with an operator. Within the scope of the present invention, the controller can alternatively be formed by non-programmable electronic components, for example an ASIC, in which the functions for carrying out the method according to the invention are implemented by circuit-technical means.

[0022] Hereinafter, embodiments of the present invention will be described in more detail with reference to the drawings. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a schematic exploded view of a cartridge for use in a rotation-based analytical method. [Diagram 2]FIG. 2 is a schematic plan view showing the heat input side of the base of the cartridge. [Diagram 3] FIG. 2 is a schematic plan view of the support plate of an analytical device in which the cartridge is intended to be used; [Figure 4] FIG. 2 is a schematic side view of the support plate of an analytical device in which the cartridge is intended to be used; [Diagram 5] FIG. 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. 13 is a schematic diagram showing the underside of the cover body of the cartridge. [Figure 8] FIG. 2 is a schematic detailed view of two chambers of a cartridge, connected by a channel, to illustrate a partial step of an analytical method. [Figure 9] FIG. 2 shows another schematic detail of two chambers of a cartridge, connected by a channel, for illustrating a partial step of an analytical method. [Figure 10] FIG. 2 shows another schematic detail of two chambers of a cartridge, connected by a channel, for illustrating a partial step of an analytical method. [Figure 11] FIG. 2 shows another schematic detail of two chambers of a cartridge, connected by a channel, for illustrating a partial step of an analytical method. [Figure 12] FIG. 3 is a diagram according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. [Figure 13] FIG. 3 is another view according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. [Figure 14] FIG. 3 is another view according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. [Figure 15] FIG. 3 is another view according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. [Figure 16] FIG. 3 is another view according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. [Figure 17] FIG. 3 is another view according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. [Figure 18] FIG. 3 is another view according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. [Figure 19] FIG. 3 is another view according to FIG. 2 showing the base of the cartridge for illustrating further partial steps of the analysis method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Corresponding parts are always provided with the same reference numbers in all the figures.

[0025] In FIG. 1, a sample container called a "cartridge" (or "disk 1" for short, due to its planar shape like a half disk) is shown in a schematic way. This disk 1 is used in a spin-based analysis method, which will be described in detail later. The disk 1 comprises a substrate 2 (also called "substrate") with a microfluidic channel and chamber structure 4. This channel and chamber structure 4 also comprises a number of chambers, which will be described in detail later, which are respectively connected to each other by assigned channels (see FIG. 2). In the unassembled state, the chambers and channels each form an "open" basin-like or groove-like recess in the substrate 2. The disk 1 therefore also comprises a sealing film 6 (or: "sealing layer"), which is heat-sealed onto the microfluidic substrate 2, thereby sealing the channel and chamber structure 4 from the side, hereinafter referred to as the "heat input side 8". 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) which is placed on the "upper side 16" of the substrate 2 (or the "underside" relative to the heat input side 8) and which is fixed to the substrate 2 in this embodiment by engaging hooks 18 (see FIG. 7) with corresponding openings 20 in the substrate 2. The cover 14 has first and second reading windows 22 and 24, respectively, through which the contents of the chambers of the substrate 2 below can be read and thereby analysed (e.g. by fluorescence detection) or at least examined.

[0026] In an optional variant (shown here), the disc 1 also comprises a (here two-part, preferably self-adhesive) label 26 attached to the cover 14. The label 26 is shaped so as to be readable through the reading windows 22 and 24. In an optional 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 thereto, so that the fluorescence can be detected without the influence of the luminescent adhesive.

[0027] 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.

[0028] The substrate 2 has a number 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 fit into these perforations 32 for positioning and fixing on a rotation plane 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).

[0029] The turntable 34 of the analysis device is used for centrifugation, i.e. for the rotation of the disk 1 around a rotation axis 40 (see FIG. 4). In this case, the turntable 34 is optionally designed to receive two disks 1 and is therefore formed with 180° rotational symmetry (see FIG. 3). Furthermore, the turntable 34 supports a number of heating elements 42, which are used for localized heating of the channels of the disk 1 and of the individual chambers of the chamber structure 4, and therefore have their outer contour adapted to the corresponding chambers. The heating elements 42 are formed in this example by resistive heating plates.

[0030] In an alternative embodiment, the heating element 42 is formed by a Peltier element, which also allows active cooling.

[0031] The individual chambers, channels and further elements of disk 1 will be explained in more detail with reference to the method sequences described below.

[0032] To carry out the analysis method, at least one disk 1 is introduced into the analysis device and positioned on the turntable 34, into which a swab 44 is inserted as a sample material carrier through the access 10 up to the swab chamber 46 of the channel and chamber structure 4, which access 10 is then closed by the capsule 12. If only one disk 1 is installed, the analysis device is configured to automatically balance the turntable 34 (in particular by arranging a counterweight on the turntable 34). For fixing, the disk 1 is sucked to the turntable 34 by means of a vacuum pump. For this purpose, the heating element 34 is provided with sealing contours 48 surrounding it. The disk 1 is applied against these and can thereby be sucked to the turntable 34 in specific areas. This allows a tight contact between the heating element 42 and the locally heated part of the disk 1.

[0033] In the initial state (i.e. without the sample or swab 44 inserted yet), the disk 1 contains pre-stored 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 in the pre-amplification chamber 56. Furthermore, primers and so-called probes (also called "genetic probes", usually in the form of poly- or oligonucleotides) are pre-stored in a number of reading chambers 58. These reading chambers 58 are visible through the reading window 24 of the cover 14. The primer pairs in the pre-amplification chambers 56 are identical or different depending on the specific purpose and / or the specific procedure of the analytical method. For example, the primers of the reading chambers 58 are pairwise identical to the primers of the pre-amplification chambers 56 or are provided for, for example, "nested" PCR ("nested" or "embedded" PCR) as known in the prior art, and are therefore made different.

[0034] The first "lyophilization chamber 60" of approximately circular shape and the second "lyophilization chamber 61" also of approximately circular shape contain pre-stored lyophilisates, which contain, for example, enzymes, polymerases, dNTPs (deoxynucleotide triphosphates), salts and / or further pre-stored reagents (for example PCR additives, nuclease inhibitors, cofactors for the enzymes involved, etc.). In this embodiment, the swab chamber 46 contains the lysis agent and the process control agent, for example spores, fungi, phages or artificial targets. The lysis chamber 62, which is connected to the swab chamber 46, contains the lysis pellet as well as a magnet and a grinding medium, for example glass and / or zirconia particles. These particles are optionally coated with EDTA, which is added to prevent coagulation when blood is used as sample material. Optionally, (activated) carbon is added to bind the inhibitors, i.e., in such an option, (activated) carbon is also pre-stored.

[0035] After the sampling, a sample carrier, in this case a swab 44, alternatively a swab 44 (also called "swab" in the medical field in sterile conditions), is inserted, for example using a blood capillary, into the disk 1, in particular into the swab chamber 46, and the access 10 is closed by the capsule 12 (see FIG. 2). 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, in particular the base 2, has a vent hole 64 arranged upstream of a filter and condensation trap 66 (in the form of a relatively small chamber). The latter allows the filter to be wetted by condensation water. However, the vent hole 64 can also be omitted in an alternative embodiment.

[0036] 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 creates a magnetic field that changes relative to the reference frame of the disk 1, causing a 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.

[0037] 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 .

[0038] Meanwhile, the turntable 34 rotates, which also rotates the disk 1, and larger sample particles are sedimented by centrifugation, thereby increasing the resistance to biochemical inhibition and reducing the risk of blockage of the microfluidic channels of the channel and chamber structure 4.

[0039] 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).

[0040] In general, however, the sample material is first homogenized by 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 additional biochemical reactions are provided in the lysis chamber 62, the reaction conditions in the lysis chamber 62 are thereby kept homogenous at the same time, i.e. a particularly stable temperature distribution is set and / or a high substance 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 can then also support subsequent amplification, since secondary structures are thereby reduced. Due to the mechanical action of the moving magnet and the forces exerted on the sample material, DNA or RNA strands can be (randomly) cut ("sheared"). The extent of shearing can then 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 here, since too strong shearing of DNA and RNA will not allow amplification.

[0041] Also in a further method step, specifically in the so-called "opening 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 as well) the rotation speed of the turntable is increased to the range of 30 Hz or more, in particular to about 60 Hz. During this centrifugation at medium to high rotation speeds, the stick pack 50 is opened in a relatively short time of the order of 5 seconds by the combination of heating and centrifugal forces. That is to say, by this heating, the tear or peel seam 67 of the stick pack 50 formed from a so-called peel film is thermally weakened. In figures 8 and 9 this process is shown diagrammatically and exemplarily for both stick packs 50 by stick pack chamber 54. Through the opening of the peel seam 67 the liquid contained in the stick pack 50 flows out into the stick pack chamber 54 (see also figure 12).

[0042] During rotation at least at 25 Hz, in particular at least 30 Hz as mentioned above, preferably at about 60 Hz, an overpressure p also builds up inside the Stickpack chamber 54 due to heating of the Stickpack chamber 54 (see FIG. 9). The overpressure p is then caused not only by the expansion of the gas contained in the respective Stickpack chamber 52 or 54 (according to the ideal gas law) but also by the vapor pressure (and thus by the sublimation of the liquid) which depends on the Stickpack liquid and the temperature in the Stickpack chamber 52 or 54. Due to the overpressure p, the liquid is forced into the channel 68 which leads to the freeze-drying chamber 61. However, due to the continued rotation of the disc 1, the centrifugal force acting in the radial direction R (perpendicular to the axis of rotation 40) opposes the liquid in the channel 68. Depending on the rotation speed and therefore on the centrifugal force, the liquid in the channel 68 can therefore rise by only a "height difference" h compared to the liquid level in the Stickpack chamber 54 (see the hatched area in FIG. 9). In this condition, an equilibrium exists between the overpressure p in the Stick-Pack chamber 54 and the “counterpressure” due to centrifugal forces in the channel 68 .

[0043] Then, when the rotation speed is reduced, the overpressure p in the stickpack chamber 54 causes a large part of the liquid (preferably more than 90%) to be expelled from the stickpack chamber 54 through the channel 68 into the freeze-drying chamber 61 (see Figures 10 and 11). This expulsion is carried out robustly against the centrifugal forces in the disk 1 even during centrifugation at 10-30 Hz. The liquid expelled from the stickpack chamber 54 dissolves the freeze-dried material placed in the freeze-drying chamber 61, which contains part of the reagents for the subsequent main amplification.

[0044] Correspondingly, the opening of the stick pack 50 in the stick pack chamber 52 and the transfer of liquid from the stick pack chamber 52 to the swab chamber 46 or to the lysis chamber 62 via the channel 68 takes place. Indeed, the lysis chamber 62 with its radially outer edge is located approximately in radial alignment with the stick pack chamber 52. However, the channel 68 leading to the lysis chamber 62 (or to the swab chamber 46) initially runs in a curved manner from the radially outer to the radially inner direction, thereby preventing a "direct" overflow of liquid in the swab chamber 46 or in the lysis chamber 62. The liquid transfer from the stick pack chamber 52 takes place before or during the above-mentioned (mechanical) lysis in the lysis chamber 62, in order to already be able to use the liquid of the stick pack 50 in the stick pack chamber 52.

[0045] In Figure 12, the open stick pack 50 is shown hatched with liquid leaking from the stick pack 50 into the stick pack chamber 54. Some liquid has already escaped from the stick pack chamber 52 into the swab chamber 46 and lysis chamber 62. Figure 13 shows the state of the stick pack chambers 52 and 54 after the liquid has been expelled from each.

[0046] In a subsequent method step, the liquid ("lysate") is transported from the lysis chamber 62 to the subsequent lyophilization chamber 60 (shown on the right above the lysis chamber 62 in Figs. 12-19), in which the lysate dissolves the lyophilized material previously stored therein (see Fig. 14). This lyophilized material may optionally contain, in addition to the above-mentioned amplification reagents, nuclease inhibitors for inactivating certain nucleases, as well as additives or cofactors such as dithiothreitol (DTT). The transport of the lysate is then carried out against the centrifugal forces due to the further rotation of the disk 1 by heating the upstream arranged 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 lyophilization chamber 60. The cooling of the subsequent chambers results in a suction effect by negative pressure, while the heating of the preceding chamber or chambers correspondingly and conversely results in the advancement of the liquid by overpressure.

[0047] The freeze-drying chamber 60 is connected to an overflow chamber 72 by an overflow channel 70. When the lysate is transferred from the lysing chamber 62 to the chamber 60, the excess lysate flows through the overflow channel 70 into the overflow chamber 72. The control chambers 74 and 76 are connected to the overflow chamber 72 and are used to check whether the disk 1 is properly filled. The lysate that flows into the overflow chamber 72 is filled from there into the control chamber 74 and into the control chamber 76 (schematically shown in FIG. 14). The filling of the control chambers 74 and 76 is used to check the proper filling of the disk 1. In particular, the filling of the control chamber 74 having an approximately square shape shown on the right side of FIG. 14, specifically the filling of the control chamber 76 connected thereto at the radially outer side, is interpreted as meaning that there is no underfilling of the disk 1. On the other hand, the filling of the approximately triangular (see FIG. 14) control chamber 74 next to the approximately square control chamber 74 on the left side, specifically the filling of the control chamber 76 connected thereto at the radially outer side, is interpreted as meaning that the disk 1 is overfilled. The total amount of liquid present here is made up of the amount of sample material introduced, typically about 105-170 microliters, and the volume of the stick pack 50 in the stick pack chamber 52, about 140-160 microliters. The filling of the corresponding control chambers 74 and 76 can be monitored continuously by a fluorescence detector through the reading window 22 of the cover 14 at a specific time point (for example at the end of the entire analytical method) or also during the filling of the lyophilization chamber 60. This makes it possible to know the time when the control chambers 74 and 76, and thus the lyophilization chamber 60, have been filled. This on the other hand also allows the estimation of possible sources of error. In an optional embodiment, a dried fluorescent dye is incorporated into the control chambers 74 and / or 76 in order to obtain a stronger signal.

[0048] 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 by means of a high rotational frequency of 40-60 Hz. As soon as the liquid state in the pre-amplification chamber 56 exceeds the opening of the respective discharge 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 arranged downstream via the assigned channel 82 (see FIG. 15).

[0049] In the next method step, preamplification is carried out in the preamplification chamber 56 under high-speed centrifugation at a rotation speed of 40-80 Hz. Overpressure in the preamplification chamber 56 and in the chamber 84 is maintained by high-speed centrifugation during preamplification. First, previously stored primers, for example spotted with trehalose, are decomposed in the preamplification chamber 56. If RNA is to be detected, it is optionally possible to first carry out a reverse transcription at a constant temperature of 35-70°C for 30 seconds to 10 or 30 minutes in order to transcribe the RNA present into DNA. However, preamplification by PCR is carried out by local and cyclical heating and cooling of the liquid in the preamplification chamber 56 in the ranges of 50-75°C and 80-100°C. The preamplification comprises 5-30 preamplification cycles. Each cycle then comprises a heating up to 80-100°C and a subsequent cooling down to 50-75°C.

[0050] The (preamplification) reaction in the preamplification chamber 56 is supported by a large convection current. This is due to the one-sided heat input from the disk 1, specifically the heat input side 8, into the preamplification chamber 56 and the simultaneous rotation. In this case, the liquid in the preamplification chamber 56 is first heated at the heat input side 8 heated by the heating element 42, whereby a heated boundary layer is formed. The density of the boundary layer is then lower than that of the remaining liquid volume. The heated liquid of the boundary layer rises in the artificial gravitational field due to the rotation of the disk 1, directed in the radial direction R, first "inward" with respect to the radial direction R and then towards the upper side 16 transverse to the radial direction R. There, the liquid cools and sinks "by gravity" outward in the radial direction R along the upper side 16 and again towards the heat input side 8. Thus, convection and flow along the radial direction R are generated by the heat input. Furthermore, the Coriolis forces also generated form a tangential flow component (i.e. perpendicular to the radial direction R in the plane direction of the disk 1), which additionally supports the mixing of the liquid. Since convection is induced by an artificial gravitational field, the faster the rotation of the disk 1, the greater the convection. Thus, the convection occurring at high rotation speeds leads to a particularly effective mixing of the reaction components in the pre-amplification chamber 56, resulting in efficient amplification conditions.

[0051] However, as a side effect, if there is a very high heat release on the unheated top side 16 of the disk 1, a large temperature gradient (e.g. 10° C. or Kelvin) can form in the liquid volume of the pre-amplification chamber 56, which can be disadvantageous. The airtight cover 14 leads to a significant reduction in the temperature gradient, most likely around 4-5 Kelvin.

[0052] In order to further reduce the heat emission, in a further embodiment, the cover 14 has a frame web 86 which surrounds the preamplification chamber 56 in a ring-like manner, thereby further reducing the heat emission due to convection at the upper side 16 (see Figs. 1 and 7). This can be omitted as an option. The frame web 86 is molded to the cover 14, i.e. is integrally connected thereto. The frame web 86 then projects towards the substrate 2 and ends at a small distance of about 100 μm from the substrate. This continuous shielding of the preamplification chambers 56 by the cover 14 and the frame web 86 allows a temperature difference of about 2 Kelvin in each preamplification chamber 56. Thus, in each preamplification chamber 56, relatively strong convection currents, in particular due to rotation, occur. However, in addition, at least in the static case, i.e. when the temperature of the heating element 42 is maintained for at least about 10 to 30 seconds, etc., a relatively high homogeneity of the reaction temperature is achieved in the preamplification chambers 56. Experience has shown that with the geometry and parameters used in this example and those described below, the static condition can be achieved already after approximately 15 seconds.

[0053] 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, high convection (and therefore relatively strong mixing) as well as uniform temperature distribution can be advantageous.

[0054] After the end of the preamplification, the rotation speed is reduced to about 5-20 Hz, in particular to about 10 Hz. This causes the amount of compressed air in the head space of the preamplification chamber 56 and in the chamber 84 to expand. This in turn results in a lowering of the liquid level in the preamplification chamber 56, since the liquid lying radially in the opening of the discharge channel 80 is at least for the most part expelled into another chamber 88 by the expanding air through the discharge channel 80. This is possible because the discharge channel 80 has a lower flow resistance, in particular a larger channel cross section, than the transfer channel 78 leading to the preamplification chamber 56.

[0055] In so doing, the disk 1 is in particular provided with a vent channel 90 which 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.

[0056] In the following method steps, the rotation speed of the disk 1 (or turntable 34) is set in the range of values ​​between 10 and 20 Hz, preferably about 15 Hz, in particular increased. Thereby, the liquid from the chamber 88 flows via a siphon 92 into a measuring chamber 94 with three radially outwardly lying "measuring fingers" or chamber projections of different volumes. These measuring fingers are then filled one after the other, whereby individual partial volumes are measured 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 the high flow resistance of these channels 96, in particular by their channel dimensions in at least one spatial direction, in particular in the cross-sectional direction, being smaller than 200 μm. The partial volumes respectively flowing through the channels 96 in the next step are therefore essentially preset by the volume of the respective upstream measuring finger. The excess liquid amount flows into a drain 98.

[0057] In the next method step (see FIG. 17), by increasing the centrifugation, i.e. increasing the rotation speed to the range of 30-80 Hz, in particular to about 60 Hz, the measured partial volumes of liquid are transferred to the respective subsequent chambers, i.e. to the freeze-drying chamber 61 shown to the left of the swab chamber 46 in FIG. 17, and further to the chamber 100.

[0058] This freeze-drying chamber 61 originally contains a "main amplification buffer" that was previously stored 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 the 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.

[0059] In the subsequent method steps, the disk 1 is rotated with relatively fast direction changes between end values ​​of -20 to 40 Hz and +20 to 40 Hz, respectively, at a rate of change of 5 to 40 Hz / s, preferably about 30 Hz / s, the signs in this case indicating the difference in the direction of rotation. The components present in the freeze-drying chamber 61 are mixed by the accelerations occurring during the direction changes and by the Euler and Coriolis forces which are generated thereby in the rotating system.

[0060] In parallel with this, the reading chambers 58, the measuring chambers 102 arranged upstream of them and the overflow chamber 104 are heated by the corresponding assigned heating elements 42. The expanded air can escape into the chamber 100 via the compensation channel 106. After the end of the mixing process, the direction change is ended and a constant rotation speed of about 20 Hz is set again. The reading chambers 58, the measuring chambers 102 and the overflow chambers 104 are then cooled again. This creates a relative negative pressure in the respective chambers 58, 102 and 104, which increases 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 force to the air pressure difference. As soon as the liquid exceeds the apex 110 of the siphon channel 108, all the liquid is transferred 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 .

[0061] In so doing, the liquid flows successively into the individual measuring chambers 102 and is thereby measured. Furthermore, the liquid in the measuring chambers 102 is firstly held back by a respective 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 in the medium rotational speed range, in particular in this case rotational speeds of about 15-25 Hz.

[0062] 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", whereby at least a large portion of the liquid is transferred to the corresponding reading chamber 58 (see Figure 19).

[0063] In a further method step, the main amplification is carried out in the reading chambers 58. For this purpose, the primers and probes respectively previously stored in the reading chambers 58 are dissolved. The dissolution of the primers and probes and the subsequent amplification are then supported by high convection in the reading chambers 58, which is caused as described above for the preamplification chambers 56. The reaction is read out in all reading chambers 58 after each cycle at about 60° C. by a fluorescence detector. The fluorescence detector detects fluorescence of different wavelengths. The reading is carried out through the reading window 24 of the cover 14. This process corresponds to the so-called “real-time PCR”. In each of the 12 reading chambers 58, multiplex reactions, for example 3-10 plex, can then be carried out. A corresponding increase in the signal of the fluorescence detector then indicates a detected target.

[0064] In order not to affect, or to affect as little as possible, the optical evaluation by the fluorescence detector, the reading chamber 58 has, radially inwardly, outside the observation area of ​​the fluorescence detector, a recess, not shown in more detail, which is used to "trap" the air bubble and prevent it from entering the observation area. An air bubble in this recess has to be deformed relatively strongly in order not to enter the observation area. Advantageously, in this embodiment, the boundary surface of the air bubble exerts a counteracting effect, being influenced in particular by the existing surface tension conditions.

[0065] Further optionally, 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 FIG. 1), thereby also reducing the heat emission from the reading chamber 58.

[0066] Instead of fluorescence detection, it is also possible to use so-called melting curve analysis, for example "high-resolution melting curve analysis" or "fast melting curve analysis", for evaluation. This allows even higher multiplexing. In an optional embodiment, in the reading chamber 58, a "real-time PCR" based on so-called intercalating dyes (for example dyes known under the brands or names "EvaGreen", "SYBR Green", "BoxTo") is carried out, in which the resulting PCR products are detected after amplification by a melting curve. In this case, up to 20 PCR products can be detected and distinguished per chamber (20-plex).

[0067] The subject matter of the invention is not limited to the examples described above. Rather, further embodiments of the invention can be derived by those skilled in the art from the above description. 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]

[0068] 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 Swab 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 chamber 61 Freeze-drying chamber 62 Melting Chamber 64 Vent hole 66 Condensate trap 67 Peel Seam 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 Chamber 102 Measurement chamber 104 Overflow Chamber 106 Compensation Channel 108 Siphon Channel 110 Vertex 112 Vent Channel 114 Valve Channel R Radial direction p Overpressure h Height difference

Claims

1. A method of operating an analytical device to perform an analytical process, comprising the steps of: Providing a cartridge (1) having a microfluidic channel and chamber structure (4) and disposing at least one film pouch (50) containing a processing liquid within a stick pack chamber (52, 54) of said cartridge (1); In an opening step, at least one of the stick pack chambers (52, 54) is heated to a temperature of 80-130°C; The method, wherein in the opening step, the cartridge (1) is rotated at a rotation speed of 20 to 80 Hz.

2. 2. The method according to claim 1, wherein as the film bag (50) a bag made of a peel film having a heat-sealed peel seam (67) is used.

3. 3. The method of claim 1 or 2, wherein the stick pack chambers (52, 54) are heated in a locally limited manner.

4. 4. The method of claim 1, wherein the volume of the stick pack chamber (52, 54) and the amount of liquid in the film bag (50) are selected such that the amount of liquid in an undrained state is about ¼ or less of the volume of the stick pack chamber (52, 54).

5. The method of claim 4, wherein a volume of about 1000 to 1500 microliters is employed for said stick pack chambers (52, 54).

6. the stick pack chambers (52, 54) are arranged radially outwardly with respect to the axis of rotation (40) of the cartridge (1) and are connected by a radially outwardly connected channel (68) to chambers (46, 61) arranged offset radially inwardly, 6. The method according to any one of claims 1 to 5, wherein the temperature remains constant and the rotation speed is reduced in order to empty the stick pack chamber (52, 54) via the channel (68) into the subsequent chamber (46, 61).

7. The method of claim 6, wherein the rotational frequency is reduced to about 5 to 20 Hz.

8. The method of claim 7, wherein the rotational speed is reduced to about 10 to 15 Hz.

9. A method according to any one of claims 1 to 8, wherein the analytical device is an analytical device for performing analytical processing by polymerase chain reaction.

10. A method of using a cartridge (1) according to any one of claims 1 to 9, comprising the steps of: The cartridge (1) comprises: A microfluidic channel-chamber structure (4) including at least one stick-pack chamber (52, 54); at least one film pouch (50) containing a processing liquid and fixedly positioned within said stick pack chamber (52, 54); A flat disc-shaped structure; A method of using a cartridge (1), wherein the stick pack chambers (52, 54) are arranged radially outward with respect to the axis of rotation (40) of the cartridge (1) and are connected to the subsequent chambers (46, 61) by channels (68) that are connected radially outward and form a curved line toward the radially inward.

11. The microfluidic channel-chamber structure (4) is at least partially covered. A method of using a cartridge (1) according to claim 10.

12. An analysis device for performing an analysis process, comprising: a receiving device for the cartridge (1); A rotary drive device that rotates the cartridge (1); a heating device (42) for heat input into said cartridge (1); An analytical device comprising: a control device configured to carry out the method according to any one of claims 1 to 9.

13. The heating device (42) provides a locally limited heat input to the cartridge (1). The analytical device of claim 12.

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