Microfluidic cartridge
Patent Information
- Authority / Receiving Office
- EP · EP
- Patent Type
- Applications
- Current Assignee / Owner
- FRIZ BIOCHEM
- Filing Date
- 2024-08-05
- Publication Date
- 2026-06-17
Smart Images
Figure EP2024072169_13022025_PF_FP_ABST
Abstract
Description
[0001] Microfluidic cartridge
[0002] The invention relates to a microfluidic cartridge for the detection of biomolecules in a sample solution and an associated detection method.
[0003] Many questions in molecular biological research and molecular diagnostics require the determination of the quantity or concentration of certain biomolecules, for example, certain nucleic acids in a sample. In this context, the term "nucleic acid" also includes nucleic acid sequences.
[0004] Powerful and rapid detection methods employ array technology using so-called DNA chips or biochips, which enable surface-sensitive detection of nucleic acid oligomer hybridization events. The actual, already highly sensitive detection is often preceded by an amplification step in which the nucleic acids to be detected are multiplied so that they are ultimately present at a concentration above the detection limit of the selected detection method.
[0005] Various methods are known in the prior art for such specific amplification of nucleic acids, for example, the polymerase chain reaction (PCR). For details and background information on PCR, reference is made to the applicant's publication DE 102011 056606 B3, the disclosure of which is incorporated into the present application.
[0006] A microfluidic cartridge for the detection of bioparticles is known from the publication DE 102015 001 998 B3. Since the described cartridge is a flow-through system in which the process fluid must be guided through the entire microchannel system, there is a risk of dissolving substances lying unprotected in the channel system. Furthermore, the double syringe used for supplying the process fluid is technically very complex as a multi-purpose tool.
[0007] Based on this, the invention is based on the object of providing a microfluidic cartridge with a simple and robust construction that allows reliable detection of an analyte in a given analysis sample.
[0008] This object is achieved by the features of the independent claims. Further developments of the invention are the subject of the dependent claims.
[0009] The invention provides a microfluidic cartridge for detecting an analyte in an analysis sample, comprising a cartridge body having a fluid interface for connecting a sample container, wherein the cartridge body comprises: a fluid reservoir for a sample receiving liquid, a main fluid channel connecting the fluid reservoir to the fluid interface and a pumping device for bidirectionally pumping fluid between the fluid reservoir and the fluid interface through the main fluid channel, an activatable closure for the main fluid channel arranged between the fluid reservoir and the fluid interface, a secondary fluid channel branching off from the main fluid channel at a branch arranged between the activatable closure and the fluid reservoir, a detection chamber for detecting the analyte using a biochip, having a fluid inlet and a fluid outlet,wherein the secondary fluid channel connects the branch from the main fluid channel to the fluid inlet of the detection chamber, a reaction chamber for amplifying the analyte to be detected in the analysis sample, with a fluid inlet and a fluid outlet, wherein the fluid outlet of the detection chamber is connected to the fluid inlet of the reaction chamber, and a dead-end pressure chamber with only one fluid inlet, which is connected to the fluid outlet of the reaction chamber.
[0010] The dead-end pressure chamber is preferably designed in a meander shape.
[0011] The secondary fluid channel advantageously has a smaller cross-section than the main fluid channel, with the cross-sectional area of the secondary fluid channel advantageously being between 10% and 90%, in particular between 30% and 60%, of the cross-sectional area of the main fluid channel. The amount of sample receiving fluid entering the secondary branch during the forward pumping step is thereby further reduced.
[0012] In an advantageous embodiment, the secondary fluid channel of the cartridge branches off from the main fluid channel at a branch angle θ that is 90° or less, in particular 70° or less, relative to the forward flow direction in the main fluid channel. The branch angle θ is advantageously as small as possible. This measure also allows the amount of sample collection fluid entering the secondary branch during the forward pumping step to be further reduced.
[0013] The fluid interface is conveniently equipped with a standardized connection, in particular a Luer-Slip or a Luer-Lock.
[0014] Advantageously, the cartridge contains a biochip whose sensor area, equipped with an array of functionalized test sites, is arranged in the detection chamber.
[0015] The pumping device of the cartridge advantageously comprises a syringe plunger slidably arranged in the fluid reservoir, which preferably has an opening for receiving a locating pin of a stepper motor. The various pumping steps can thus be precisely controlled. The fluid reservoir and the syringe plunger are expediently cylindrical.
[0016] In an advantageous embodiment, the activatable closure is formed by a valve area and a rubber bead that is inserted into a ball channel extending perpendicularly from the main fluid channel. To fire (i.e., activate) the closure, the rubber bead can be pressed into the valve area.
[0017] The cartridge body is advantageously formed from an injection-molded body, for example from polycarbonate or polypropylene, which is sealed on its top or bottom side with a cover film.
[0018] The reaction chamber is advantageously configured for performing PCR and / or RT-PCR (reverse transcription polymerase chain reaction). The abbreviation (RT-)PCR refers to both process variants in this description.
[0019] The ratio of the volume of the pressure chamber to the volume of the rest of the fluidic system located outside the fluid reservoir is preferably between 2:1 and 1:2, particularly preferably approximately 1:1. By filling the fluidic system, with the exception of the pressure chamber, with the analysis sample, the air-filled initial volume is reduced to 2 / 3 to 1 / 3, in particular to approximately half, thus increasing the pressure in the pressure chamber by a factor of 1.5 to 3, in particular by a factor of approximately 2. This provides a counterpressure to suppress unwanted gas bubbles during subsequent processing, in particular when carrying out (RT-)PCR.
[0020] The invention also includes a method for detecting an analyte in an analysis sample using a microfluidic cartridge of the type described, in which the cartridge is connected to a sample container containing an analyte to be examined, in a forward pumping step, the sample receiving liquid is pumped from the fluid reservoir into the sample container using the pumping device, in a reverse pumping step, at least a portion of the sample receiving liquid is pumped back into the fluid reservoir together with the analyte as the analysis sample, the activatable closure is activated and the main fluid channel is thereby decoupled from the fluid interface, in an internal forward pumping step, a portion of the analysis sample is pumped from the fluid reservoir into the detection chamber and the reaction chamber, the analyte is processed, in particular amplified, in the reaction chamber,In an internal reverse pumping step, a portion of the analysis sample with the processed analyte is pumped into the detection chamber, and the analyte is detected in the detection chamber using a biochip.
[0021] Advantages of the invention as well as further embodiments are explained below with reference to the figures, in which a true-to-scale and true-to-proportion reproduction has been omitted in order to increase clarity.
[0022] They show:
[0023] Fig. 1 schematically shows a microfluidic cartridge according to an embodiment of the invention and a sample container with a swab sample to be examined,
[0024] Fig. 2 to 6 Intermediate steps in the process flow for the detection of biomolecules in the swab sample with the microfluidic cartridge of Fig. 1, and
[0025] Fig. 7 schematically shows a microfluidic cartridge according to another embodiment of the invention. The invention will now be explained using microfluidic cartridges for the detection of specific nucleic acids in an analysis sample.
[0026] Figure 1 schematically shows a microfluidic cartridge 20 that can be connected to a sample container 10 via a fluid interface 30. In the exemplary embodiment, the sample container is a swab tube 10 with a hollow swab rod 12, at the distal end of which a cotton ball 14 carries a swab sample 50 from a patient, which is to be microbiologically examined, for example, for MRS A strains (methicillin-resistant Staphylococcus aureus).
[0027] It is understood that other microbiological or other molecular diagnostic tests can also be performed with the cartridge 20. Other types of sample containers, such as a sputum cup, can also be connected to the cartridge 20. For this purpose, the fluid interface 30 can be equipped with a standardized connector, for example, a Luer slip or a Luer lock, which can be connected to a corresponding counterpart on the sample container side.
[0028] The microfluidic cartridge 20 has a cartridge body 22, which consists, for example, of an injection-molded body made of polycarbonate or polypropylene sealed with a cover film and in which the fluid interface 30 and the other elements of the fluidic system 24 of the cartridge 20, described in more detail below, are formed.
[0029] As a special feature, the fluidic system 24 consists of a main branch 26 and a secondary branch 28 designed as a dead-end. The main branch 26 comprises a fluid reservoir 32 for receiving a sample receiving liquid 52, a main fluid channel 34 that connects the fluid reservoir 32 to the fluid interface 30, and a pumping device 36 for bidirectionally pumping fluid between the fluid reservoir 32 and the fluid interface 30 through the main fluid channel 34.
[0030] An activatable closure 38 is arranged between the fluid reservoir 32 and the fluid interface 30, with which the main fluid channel 34 can be closed. In the delivery state of the cartridge 20 shown in Fig. 1, the closure 38 is in its open state.
[0031] The secondary branch 26 of the fluidic system 24 contains a secondary fluid channel 40, which branches off from the main fluid channel 34 at a branch 42 arranged between the activatable closure 38 and the fluid reservoir 32, a detection chamber 44, a reaction chamber 46 and a dead-end pressure chamber 48.
[0032] The detection chamber 44 serves to detect an analyte contained in an analysis sample by means of a biochip 54 inserted into the cartridge 20. The detection chamber 44 has a fluid inlet 44-1 and a fluid outlet 44-0, wherein the fluid secondary channel 40 connects the branch 42 from the main fluid channel 34 to the fluid inlet 44-1 of the detection chamber 44.
[0033] The reaction chamber 46 serves to amplify the analyte to be detected, for example, by means of (RT-)PCR, and has a fluid inlet 46-1 and a fluid outlet 46-0, wherein the fluid outlet 44-0 of the detection chamber is connected to the fluid inlet 46-1 of the reaction chamber 46. The pressure chamber 48 is designed as a dead-end chamber and contains only a fluid inlet 48-1, but no fluid outlet. The fluid inlet 48-1 of the dead-end pressure chamber 48 is connected to the fluid outlet 46-0 of the reaction chamber 46. The channel pieces connecting the detection chamber 44 and the reaction chamber 46, or the reaction chamber 46 and the pressure chamber 48, are also considered part of the secondary fluid channel 40.
[0034] The fluid secondary channel 40 has, in particular, a smaller cross-section than the fluid main channel 34. In addition, the branch angle 0 of the fluid secondary channel 40 to the forward flow direction 70 (Fig. 2) of the fluid main channel is 90° or less.
[0035] The basic process flow for detecting biomolecules with the microfluidic cartridge 20 is, for example, as follows with reference to Figures 2 to 6:
[0036] First, the cartridge 20 is placed onto the sample container 10 containing the swab sample 50 to be analyzed, establishing a fluid connection between the fluid reservoir 32 and the sample container 10. In the as-delivered state of the cartridge, the fluid interface 30 may be protected, for example, by a Luer cap, which is removed before the cartridge is attached. The fluid reservoir 32 may also be sealed, for example, with a wax seal, which is melted by heating before the analysis begins, thereby opening the fluid connection between the fluid reservoir 32 and the sample container 10. Furthermore, wax-protected lysis chemicals (not shown) may be placed between the fluid reservoir 32 and the fluid interface 30, which are exposed by melting the wax protection.
[0037] Referring now to Fig. 2, after these preparatory steps, the sample receiving liquid 52 provided in the fluid reservoir 32, together with the exposed lysis chemistry, is pumped through the main fluid channel 34 via the fluid interface 30 into the sample container 10 in a forward pumping step 70. In a specific embodiment, for example, 400 μl of sample receiving liquid 52 can be provided and pumped into the swab tube in the forward pumping step. To achieve a defined end state, the pumping process can be continued until the escape of air indicates the complete emptying of the fluid reservoir 32.
[0038] In the forward pumping step 70, a small amount of sample receiving liquid 52 also reaches the secondary branch 28 via the branch 42. However, this amount is negligible due to the geometry of the branch 42 and the fluid channels 34, 40, and due to the dead-end design of the secondary branch 28. Therefore, in the forward pumping step 70, the sample receiving liquid 52 is pumped almost exclusively into the sample container 10, as shown in Fig. 2.
[0039] The swab area containing the swab sample 50 can be additionally heated, subjected to ultrasound, and / or subjected to mechanical shear forces or other mechanical measures to promote the solubilization, lysis, and / or inactivation of the swab sample. After lysis 60 of the swab sample 50, the sample collection fluid contains the analyte 58 to be detected contained in the swab sample 50, as shown in the bottom left of Fig. 2, thus forming the analysis sample 62.
[0040] After lysis 60 has taken place, the analysis sample 62 is pumped back into the fluid reservoir 32 in a reverse pumping step 72, as shown in Fig. 3. In the exemplary embodiment, for example, approximately 300 μl of analysis sample 62 can be pumped back into the fluid reservoir 32, the remainder of the analysis sample (« 100 μl) remains in the cotton swab 14 of the swab stick.
[0041] After the reverse pumping step 72, the activatable closure 38 is activated to close the fluid connection between the fluidic system 24 of the cartridge 20 and the fluid interface 30. After the closure of the closure 38, in the exemplary embodiment, approximately 300 μl of analysis sample 62 and approximately 700 μl of air are present in the fluid reservoir 32.
[0042] Referring now to Fig. 4, after closing the main fluid channel 34, the analysis sample 62 is pumped into the secondary branch 28 of the fluidic system 24 in an internal forward pumping step 74 until the detection chamber 44 and the reaction chamber 46 are filled with the analysis sample 62.
[0043] Since the secondary branch 28 is designed as a dead-end with a distal dead-end pressure chamber 48, a counterpressure of approximately 2 bar builds up during the internal forward pumping step 74 due to the reduction in the air volume in the pressure chamber 48, which leads to the suppression of unwanted gas bubbles during the subsequent heating of the analysis sample 62 in the reaction chamber 46. In the exemplary embodiment, the reaction chamber 46 has, for example, a receiving volume of approximately 25 pl, the detection chamber a receiving volume of only approximately 1-2 μl, and the pressure equalization chamber a receiving volume of approximately 35 pl. The various sections of the main fluid channel 34 and the secondary fluid channel 40 together have a receiving volume of approximately 10 pl.
[0044] By completely filling the reaction chamber 46 and the various sections of the main fluid channel and the secondary fluid channel with the analysis sample 62 according to Fig. 4, the initial volume of approximately 70 μl, which is initially filled with air, is reduced to approximately half, namely the volume of the pressure chamber (approximately 35 pl). The pressure in the pressure chamber is thereby doubled to approximately 2 bar. Such a counterpressure of approximately 2 bar has proven to be very effective for suppressing unwanted gas bubbles during the subsequent performance of an (RT-)PCR. However, with appropriate selection of the volumes, it is also readily possible to generate a different counterpressure in the pressure chamber, for example, 1.5 bar, 2.5 bar, or 3 bar.
[0045] After the reaction chamber 46 has been filled, a (RT) polymerase chain reaction (PCR) is carried out there to amplify the nucleic acids of the analyte 58 to be detected. The chemicals required for this are pre-packaged in the reaction chamber 46, for example, protected under a layer of wax. To start the amplification, the wax layer is melted by increasing the temperature, and the PCR chemicals are thereby added to or released from the analysis sample 62 in the reaction chamber 46. During the PCR, the molten wax remains liquid or is absorbed by one or more wax reservoirs in the edge region of the reaction chamber 46 to ensure that no solidified wax clogs adjacent fluid channels. This can be exploited by the fact that liquid wax is lighter than the surrounding liquid and therefore rises to the top.In the reaction chamber 46, bulges can therefore be provided at predetermined locations in the edge region, in which the rising liquid wax collects when the cartridge 20 is positioned at an appropriate angle during the (RT-)PCR is carried out.
[0046] The cyclic temperature profile required for the PCR in the reaction chamber 46 is generated, for example, with the aid of a heating / cooling device of the analysis device, whereby a controlled process of the (RT-)PCR is ensured by the counterpressure generated by the dead-end pressure chamber 48.
[0047] After the amplification of the analyte 58 in the reaction chamber 46, an amplified analysis sample 64 is present in the reaction chamber 46, in which the analyte 58 to be detected is present in a concentration many times higher than before the (RT-)PCR step, as schematically shown in Fig. 5.
[0048] For the detection step, a small portion of the amplified analysis sample 64 is now transferred into the detection chamber 44 in an internal reverse pumping step 76, as illustrated in Fig. 6.
[0049] The sensor area of the inserted biochip 54 is arranged in the detection chamber 44, with an array of functionalized test sites 56 for the electrochemical detection of nucleic acids in the amplified analysis sample 64. During electrochemical detection, the test sites 56 each generate an electrical signal, the magnitude of which represents a measure of the nucleic acid concentration in the transferred detection volume. These electrical signals are then picked up at contact pads 58 of the biochip and evaluated in the analysis device.
[0050] In a pure endpoint determination, (RT-)PCR provides a qualitative yes / no answer as a result, i.e. a statement as to whether the analyte to be detected is present in the analysis sample or not.
[0051] However, the microfluidic cartridge 20 according to the invention also allows for the performance of quantitative or semi-quantitative (RT-)PCR. For this purpose, the amount of nucleic acid present in the sample solution is determined while the reaction is running, enabling a more precise determination of the nucleic acid concentration in the analysis sample than with a mere endpoint determination at the end of an (RT-)PCR.
[0052] The steps illustrated in Figs. 5 and 6 of amplification in the reaction chamber 46 and the transfer of a detection volume into the detection chamber 44 are repeated several times. For example, as described in connection with Fig. 5, a first predetermined number of cycles of the amplification reaction, for example, 10 cycles, is first carried out in order to raise the concentration of nucleic acid in the analysis sample to the measurement range.
[0053] Subsequently, with the aid of the pump device 36, a defined volume of analysis sample containing amplified nucleic acids is transferred from the reaction chamber 46 to the detection chamber 44, and the concentration of the nucleic acids is determined by electrochemical detection. Subsequently, a further PCR cycle is repeatedly performed in order to further increase the concentration of nucleic acids in the reaction chamber 46. For example, after every 5 cycles, a defined volume of sample solution containing amplified nucleic acids is again transferred from the reaction chamber 46 to the detection chamber 44, and the concentration of nucleic acids is determined electrochemically. In this way, for example, a total of 30 to 40 PCR cycles can be performed, and concentration values can be determined after 10, 15, 20, 25, 30, and possibly 35 and 40 cycles.With such an approach, a Ct value can be specified (semi-)quantitatively, which indicates after which number of cycles a predetermined threshold concentration is exceeded.
[0054] When performing (semi-)quantitative (RT-)PCR, two variants are considered: in a first variant, the volume removed from the reaction chamber is pumped back after each detection step, while in a second variant, no pumping back step takes place and therefore the amount of analysis fluid in the reaction chamber decreases with each detection step.
[0055] Figure 7 shows a further embodiment of a microfluidic cartridge 120 according to the invention with an injection-molded cartridge body 122 and a fluid interface 130 formed in the cartridge body for connection to a sample container, such as the swab tube 10 of Fig. 1.
[0056] A fluidic system 124 with a main branch 126 and a secondary branch 128 configured as a dead-end is formed in the cartridge body 122. The main branch 126 comprises a cylindrical fluid reservoir 132 for receiving a sample receiving liquid 52, a main fluid channel 134 connecting the fluid reservoir 132 to the fluid interface 130, and a pumping device 136 for bidirectionally pumping fluid between the fluid reservoir 132 and the fluid interface 130 through the main fluid channel 134.
[0057] To pump controlled fluid volumes, the pumping device 136 can be controlled by a stepper motor of an analysis device (not shown in the figures). For this purpose, the pumping device 136 comprises a syringe plunger 150 slidably arranged in the fluid reservoir cylinder 132, with a circular opening 152 at the plunger end, into which a dowel pin of the stepper motor can engage in order to carry out the various forward and reverse pumping steps 70, 72, 74, 76 in a precisely controlled manner.
[0058] Between the fluid reservoir 132 and the fluid interface 130, an activatable closure 138 is arranged, which in the embodiment of Fig. 7 is formed by a valve region 160 and a rubber ball 162, which is pre-inserted in a ball passage extending perpendicularly from the main fluid channel 34.
[0059] In the open state of the closure 138, the rubber bead 162 is clamped in the ball path outside the main fluid channel 134. To close the closure 138, the rubber bead 162 can be pressed into the valve area 160 with a plunger, where it completely closes the main fluid channel 134.
[0060] The secondary branch 126 of the fluidic system 124 contains a secondary fluid channel 140, which branches off from the main fluid channel 134 at a branch 142 arranged between the activatable closure 138 and the fluid reservoir 132, a detection chamber 144, a reaction chamber 146 and a dead-end pressure chamber 148. As described in Fig. 2, the said chambers are fluidically connected to one another via the secondary fluid channel 140 and can in particular have the receiving volumes described in connection with Figs. 2 to 6.
[0061] As shown in Fig. 7, the secondary fluid channel 40 has a significantly smaller cross-section than the main fluid channel 34. Furthermore, the branch angle 0 of the secondary fluid channel 40 relative to the forward flow direction of the main fluid channel is only approximately 75°. Both measures contribute to ensuring that hardly any sample receiving fluid reaches the secondary branch 128 during the forward pumping step 70.
[0062] The cartridge 120 is shown in Fig. 7 together with the inserted biochip 54 for the detection of an analyte in the detection chamber 144.
[0063] PCR chemicals protected by a meltable wax cover are placed in the reaction chamber 146 at three locations 170. Furthermore, the reaction chamber 146 has a wax collection reservoir 172 in which the melted wax from the wax cover can collect during analysis without clogging the narrow fluid side channel 40.
[0064] The dead-end pressure chamber 148 is designed in a meander shape, which has proven particularly useful in practice for building up a counterpressure for bubble suppression.
[0065] The process sequence when using the cartridge 120 with the forward and reverse pumping steps 70, 72, 74, 76 essentially corresponds to the process sequence described above.
Claims
Patent claims 1. A microfluidic cartridge for detecting an analyte in an analysis sample, comprising a cartridge body having a fluid interface for connecting a sample container, wherein the cartridge body comprises: a fluid reservoir for a sample receiving liquid, a main fluid channel connecting the fluid reservoir to the fluid interface, and a pumping device for bidirectionally pumping fluid between the fluid reservoir and the fluid interface through the main fluid channel, an activatable closure for the main fluid channel arranged between the fluid reservoir and the fluid interface, a secondary fluid channel branching off from the main fluid channel at a branch arranged between the activatable closure and the fluid reservoir, a detection chamber for detecting the analyte by means of a biochip, having a fluid inlet and a fluid outlet,wherein the secondary fluid channel connects the branch from the main fluid channel to the fluid inlet of the detection chamber, a reaction chamber for amplifying the analyte to be detected in the analysis sample, with a fluid inlet and a fluid outlet, wherein the fluid outlet of the detection chamber is connected to the fluid inlet of the reaction chamber, and a dead-end pressure chamber with only one fluid inlet, which is connected to the fluid outlet of the reaction chamber.
2. Microfluidic cartridge according to claim 1, characterized in that the dead-end pressure chamber is designed in a meander shape.
3. Microfluidic cartridge according to claim 1 or 2, characterized in that the fluid secondary channel has a smaller cross-section than the fluid main channel, wherein the cross-sectional area of the fluid secondary channel is advantageously between 10% and 90%, in particular between 30% and 60% of the cross-sectional area of the fluid main channel.
4. Microfluidic cartridge according to at least one of claims 1 to 3, characterized in that the secondary fluid channel branches off from the main fluid channel at a branching angle which, relative to the forward flow direction in the main fluid channel, is 90° or less, in particular 70° or less.
5. Microfluidic cartridge according to at least one of claims 1 to 4, characterized in that the fluid interface is equipped with a standardized connection, in particular a Luer slip or a Luer lock.
6. Microfluidic cartridge according to at least one of claims 1 to 5, characterized in that the cartridge contains a biochip whose sensor region equipped with an array of functionalized test sites is arranged in the detection chamber.
7. Microfluidic cartridge according to at least one of claims 1 to 6, characterized in that the pumping device comprises a syringe piston which is arranged displaceably in the fluid reservoir and which is preferably which has an opening for receiving a dowel pin of a stepper motor.
8. Microfluidic cartridge according to claim 7, characterized in that the fluid reservoir and the syringe plunger are cylindrical.
9. Microfluidic cartridge according to at least one of claims 1 to 8, characterized in that the activatable closure is formed by a valve region and a rubber bead which is pre-inserted in a ball channel extending perpendicularly away from the main fluid channel.
10. Microfluidic cartridge according to at least one of claims 1 to 9, characterized in that the cartridge body is formed from an injection-molded body sealed with a cover film.
11. Microfluidic cartridge according to at least one of claims 1 to 10, characterized in that the reaction chamber is designed to carry out an (RT-)PCR.
12. Microfluidic cartridge according to at least one of claims 1 to 9, characterized in that the ratio of the volume of the pressure chamber to the volume of the rest of the fluidic system located outside the fluid reservoir is between 2:1 and 1:2, advantageously approximately 1:
1.
13. A method for detecting an analyte in an analysis sample using a microfluidic cartridge according to one of claims 1 to 12, in which the cartridge is connected to a sample container with an analyte to be examined, in a forward pumping step the sample receiving liquid is pumped from the fluid reservoir into the sample container using the pumping device, in a backward pumping step at least a portion of the sample receiving liquid is pumped back into the fluid reservoir together with the analyte as the analysis sample, the activatable closure is activated and the main fluid channel is thereby decoupled from the fluid interface, in an internal forward pumping step a portion of the analysis sample is pumped from the fluid reservoir into the detection chamber and the reaction chamber, the analyte is processed, in particular amplified, in the reaction chamber, in an internal backward pumping step a portion of the analysis sample with the processed analyte is pumped into the detection chamber, and the analyte is detected in the detection chamber using a biochip.