Microfluidic device and method for analyzing a sample of biological material

Abstract

Microfluidic device (100) for analyzing a sample of biological material, wherein the microfluidic device (100) has the following features: a first reaction chamber (110) which is designed to carry out a first polymerase chain reaction on the sample in order to produce a first reaction product; a mixing chamber (130) fluidically connected or connectable to the first reaction chamber (110), which is configured to mix the first reaction product with a dilution medium to produce a dilute mixture; and at least one second reaction chamber (150) fluidically connected or connectable to the mixing chamber (130), which is designed to carry out a second polymerase chain reaction on the diluted mixture for the purpose of analyzing the sample.

Description

State of the art

[0001] The present invention relates to a microfluidic device for analyzing a sample of biological material, further to a method for analyzing a sample of biological material, further to a device for carrying out the steps of the method and to a corresponding computer program.

[0002] A microfluidic diagnostic system, such as a so-called lab-on-chip (LoC), enables the miniaturized and integrated implementation of complex fluidic workflows, particularly for the specific detection of a wide variety of molecules. US 2011 / 0070578 A1 discloses a DNA analysis device that performs a polymerase chain reaction and dilution for subsequent electrophoretic separation. Disclosure of the invention

[0003] Against this background, the approach presented here presents a microfluidic device for analyzing a sample of biological material, a method for analyzing a sample of biological material, a device using this method, and finally a corresponding computer program according to the main claims. Advantageous embodiments emerge from the respective subclaims and the following description.

[0004] According to embodiments of the invention, in particular, a nested polymerase chain reaction (PCR) can be created in which a sample is diluted between a first and a second polymerase chain reaction, which can be referred to as a "diluted nested PCR." Thus, according to embodiments of the invention, microfluidic structures and process flows can be provided for performing a nested polymerase chain reaction with dilution and using identical primer sequences in the first and second polymerase chain reactions.

[0005] Advantageously, according to embodiments of the invention, in particular, implementation of PCR assays without primer modification can be enabled, for example, by using dUTP-containing (dUTP = deoxyuridine triphosphate) primers, primers with different melting temperatures, "capture primers" used in the second PCR, which are complementary to the primers of the first PCR and thus deactivate the primers, or the like. Thus, in particular, there is no need to resort to the use of dUTP-containing primers in the first PCR, whereby, after transferring the PCR product to the singleplex reaction chambers, an UNG digestion (UNG = uracil-N-glycosylase) would be performed to deactivate the primers of the first reaction. Therefore, special nucleotides in primer synthesis and an additional enzyme system for UNG digestion can be dispensed with.Likewise, there is no need to use primers with different melting temperatures. For example, primers with a melting temperature between 55 and 65°C would be used in the first multiplex PCR, while primers with a melting temperature of around 72°C would be used in the second singleplex PCR. Thus, PCR primers for the first and second PCR do not need to be designed in different lengths for different melting temperatures.

[0006] Therefore, according to embodiments of the invention, costs and effort can be saved in the nested polymerase chain reaction. This can also increase the flexibility of a LoC system, as many different PCR assays or biocontents can be implemented in a LoC without adaptation. For example, there are significantly more assays in which DNA molecules are detected directly via a singleplex to quadplex PCR reaction than in a combination of multiplex PCR plus subsequent microarray analysis.

[0007] The advantages of nested polymerase chain reactions are that pre-amplification of nucleic acid molecules contained in the sample in the first polymerase chain reaction can generate sufficient material for individual detection reactions in the second polymerase chain reaction. This prevents, for example, a sample material to be analyzed that contains very few DNA molecules from being distributed directly across multiple reaction chambers, resulting in the individual chambers containing too little genetic starting material for a PCR. Rather, by increasing the quantity of nucleic acid molecules to be analyzed in the first PCR, each desired nucleic acid molecule can be detected in each reaction chamber of the second PCR. By nesting two polymerase chain reactions, a very large number of genetic traits can be detected from a small sample volume in singleplex reactions.

[0008] Furthermore, no DNA microarray needs to be used to detect different DNA molecules. This also reduces the resolution requirements for an optical detection unit and shortens processing time. Furthermore, real-time capability can be provided according to embodiments of the invention. Reactions can be carried out in triplicate, for example, which can increase the significance of the reactions.

[0009] A microfluidic device for analyzing a sample of biological material is presented, wherein the microfluidic device has the following features: a first reaction chamber configured to perform a first polymerase chain reaction on the sample to produce a first reaction product; a mixing chamber fluidically connected or connectable to the first reaction chamber, which is designed to mix the first reaction product with a dilution medium to produce a diluted mixture; and at least one second reaction chamber fluidically connected or connectable to the mixing chamber, which is designed to carry out a second polymerase chain reaction on the diluted mixture for analyzing the sample.

[0010] The microfluidic device can be used, for example, in conjunction with analytical systems, in particular for microfluidic lab-on-chip systems for environmental analysis or medical diagnostics. The microfluidic device can be a microfluidic system or an analytical device, in particular a microfluidic lab-on-chip or chip laboratory for medical diagnostics, microbiological diagnostics, or environmental analysis. The sample can be a liquid to be analyzed, typically a liquid or liquefied patient sample, e.g., blood, urine, stool, sputum, cerebrospinal fluid, lavage, a rinsed swab, or a liquefied tissue sample, or a sample of a non-human material. Cells contained in the sample can be, for example, human cells, e.g., blood cells or the like, animal cells, or pathogenic cells or pathogens, e.g., viruses or microorganisms, such as, for example,Bacteria or fungi, which contain DNA and / or RNA, i.e., nucleic acid molecules. The reaction chambers can be fluidly connected to the mixing chamber, for example, using valves.

[0011] According to one embodiment, at least one substance for carrying out the first polymerase chain reaction can be stored upstream in the first reaction chamber. The dilution medium can be stored upstream in the mixing chamber. At least one substance for carrying out the second polymerase chain reaction can be stored upstream in the at least one second reaction chamber and additionally or alternatively in the mixing chamber. According to one embodiment, the device can have at least two or at least three second reaction chambers. Such an embodiment offers the advantage of enabling a particularly space-saving arrangement of chambers in the microfluidic device and a simplification of the arrangement of chambers. According to one embodiment, the reaction mix can also be introduced into the reaction chamber in a ready-to-use state.

[0012] A storage chamber for storing dilution medium can also be provided, which is fluidically connected or connectable to the mixing chamber. In this case, at least one substance for carrying out the first polymerase chain reaction can be stored upstream in the first reaction chamber, wherein the dilution medium can be stored upstream in the storage chamber. In this case, at least one substance for carrying out the second polymerase chain reaction can be stored upstream in the at least one second reaction chamber, in the storage chamber, and additionally or alternatively in the mixing chamber. Such an embodiment offers the advantage that a variable mixing ratio or a variable dilution can be generated in the mixing chamber. According to one embodiment, the reaction mix can also be introduced into the reaction chamber already "ready-to-use."

[0013] In particular, a volume of the first reaction chamber and a volume of the mixing chamber can be designed to bring about a mixing ratio in the diluted mixture that is suitable for at least partially excluding primers from the first polymerase chain reaction from the second polymerase chain reaction. By diluting and the associated reduction in the concentration of primers from the first polymerase chain reaction, participation of these primers in the second polymerase chain reaction is suppressed. A volume of the storage chamber can also be designed to bring about a mixing ratio in the diluted mixture that is suitable for at least partially inactivating primers from the first polymerase chain reaction for the second polymerase chain reaction. In the case of a nested PCR, PCR primers from the first and second polymerase chain reaction can thus be used.multiplex polymerase chain reaction can be essentially excluded from the subsequent singleplex polymerase chain reaction. Therefore, it is possible to detect a specific target molecule. In other words, the PCR product of the first PCR can be diluted to such an extent that the likewise diluted primers of the first PCR are essentially at least partially effectively inactivated in the second PCR. The biochemical background of the described approach is that the concentration of the primers of the first PCR is reduced or decreased by diluting the reaction volume (after completion of the first PCR) to such an extent that these primers can no longer actively participate in the second reaction in the subsequent second PCR because the concentration of these primers is insufficient for this.The reaction volume of the first PCR is then distributed over several chambers containing primers for the second PCR, which mix with the diluted mix of the first PCR when the chambers are filled, resulting in functional reaction mixes.

[0014] Furthermore, a ventilation opening can be provided, which is formed in the mixing chamber as a compensating opening relative to an ambient pressure. The ventilation opening can be designed to enable pressure equalization between the mixing chamber and the environment of the microfluidic device. Such an embodiment offers the advantage that excess air in the mixing chamber can escape through the ventilation opening, thus facilitating the mixing process in the mixing chamber.

[0015] According to one embodiment, a pump chamber fluidically connected between the first reaction chamber and the mixing chamber and a plurality of valves can be provided. A plurality of second reaction chambers can also be provided. The first reaction chamber can be arranged between a first pair of valves, the pump chamber can be arranged between a second pair of valves, and the plurality of second reaction chambers can be arranged between a plurality of third pairs of valves. The chambers of the microfluidic device can be fluidically connected by means of channels, wherein the valves can be configured to influence fluid flow through the channels.

[0016] The storage chamber can be fluidically connected to the mixing chamber via a channel and a valve. Such an embodiment offers the advantage of providing a particularly efficient, space-saving, and structurally simple microfluidic device.

[0017] A method for analyzing a sample of biological material is also presented, wherein the method can be carried out using a microfluidic device having a first reaction chamber, a mixing chamber fluidically connected to the first reaction chamber, and at least one second reaction chamber fluidically connected to the mixing chamber, the method comprising the following steps: performing a first polymerase chain reaction on the sample in the first reaction chamber to produce a first reaction product; Mixing the first reaction product with a dilution medium in the mixing chamber to produce a diluted mixture; and Performing a second polymerase chain reaction on the diluted mixture in the at least one second reaction chamber to analyze the sample.

[0018] The method can advantageously be carried out in conjunction with an embodiment of the aforementioned microfluidic device in order to analyze a sample of biological material. Furthermore, the method can comprise, between the carrying out step and the mixing step, a first step of conveying the first reaction product from the first reaction chamber into the mixing chamber, and, between the mixing step and the carrying out step, a second step of conveying the diluted mixture from the mixing chamber into the at least one second reaction chamber. The conveying steps can be carried out by controlling at least one valve and, additionally or alternatively, at least one conveying device. The at least one valve and / or the at least one conveying device can comprise interfaces designed to receive control signals.

[0019] According to one embodiment, control signals for controlling at least one valve and, additionally or alternatively, at least one conveying device of the microfluidic device can be generated in the mixing step in order to achieve a mixing ratio in the diluted mixture that is suitable for excluding primers from the first polymerase chain reaction from the second polymerase chain reaction. Such an embodiment offers the advantage of enabling precise detection of nucleic acid sequences while avoiding the use of specific or different primers.

[0020] In a nested PCR, two polymerase chain reactions can be carried out consecutively. In the first polymerase chain reaction, several primer pairs can be used, allowing multiple nucleic acid sequences or, in particular, DNA sequences to be specifically amplified simultaneously in one reaction chamber in the sense of a multiplex PCR. A PCR product from the first reaction can then be distributed across several spatially separated reaction chambers, each of which can contain a primer pair. This allows amplified nucleic acid molecules or, in particular, DNA molecules from the first PCR to act as so-called "template DNA" in the second PCR. This allows one nucleic acid molecule to be detected in each chamber in the second PCR in the sense of a singleplex PCR. First, a sample solution to be examined can be introduced, for example, into a first reaction chamber and pre-amplified using multiplex PCR.In this reaction, several different segments of a nucleic acid can be amplified, for example, between 5 and 100. After mixing or dilution, this PCR product can be distributed, for example, into many second reaction chambers, in which substances such as polymerase and a primer pair can be pre-stored. This allows different nucleic acid molecules to be detected in individual singleplex PCR reactions in parallel or highly parallel fashion, particularly in the individual chambers.

[0021] The approach presented here further provides a device designed to perform, control, or implement the steps of a variant of a method presented here in corresponding devices. This embodiment of the invention in the form of a device also allows the problem underlying the invention to be solved quickly and efficiently.

[0022] In this case, a device can be understood as an electrical device that processes sensor signals and outputs control and / or data signals depending on them. The device can have an interface, which can be implemented in hardware and / or software. In a hardware implementation, the interfaces can, for example, be part of a so-called system ASIC, which contains a wide variety of functions of the device. However, it is also possible for the interfaces to be separate integrated circuits or to consist at least partially of discrete components. In a software implementation, the interfaces can be software modules that are present, for example, on a microcontroller alongside other software modules.

[0023] Also advantageous is a computer program product with program code that can be stored on a machine-readable medium such as a semiconductor memory, a hard disk memory or an optical memory and is used to carry out and / or control the steps of the method according to one of the embodiments described above, in particular when the program product is executed on a computer or a device.

[0024] The approach presented here is explained in more detail below using the attached drawings. They show: Fig. 1 is a schematic representation of a microfluidic device according to an embodiment of the present invention; Fig. 2 is a schematic representation of a microfluidic device according to an embodiment of the present invention; Fig. 3 and Fig. 4 Comparative representations of analysis results according to embodiments of the present invention; and Fig. 5 a flowchart of a method according to an embodiment of the present invention.

[0025] In the following description of advantageous embodiments of the present invention, the same or similar reference numerals are used for the elements shown in the various figures and having a similar effect, whereby a repeated description of these elements is omitted.

[0026] Fig. Figure 1 shows a schematic representation of a microfluidic device 100 for analyzing a sample of biological material according to an embodiment of the present invention. The microfluidic device 100 is embodied, for example, as a microfluidic chip or a pocket-sized laboratory or LoC (lab on chip), in particular for medical diagnostics, microbiological diagnostics, or environmental analysis. The microfluidic device 100 is shown in Fig. 1 here a first reaction chamber 110, a pumping chamber 120, a mixing chamber 130, a ventilation opening 140, a plurality of second reaction chambers 150, wherein in Fig. 1 shows only four second reaction chambers 150 by way of example, as well as a plurality of valves 161, 162, 163, 164, 165 and 166.

[0027] The first reaction chamber 110 is arranged between a first valve 161 and a second valve 162. The first valve 161 is arranged in a microfluidic supply channel. The sample can be supplied to the first reaction chamber 110 via the microfluidic supply channel. The first reaction chamber 110 is designed to perform a first polymerase chain reaction on the sample to generate a first reaction product. The second valve 162 is arranged in a first microfluidic connecting channel. The first reaction chamber 110 is fluidically connected to the pump chamber 120 via the first microfluidic connecting channel.

[0028] The pump chamber 120 is arranged between a third valve 163 and a fourth valve 164. The third valve 163 is arranged in the first microfluidic connection channel. Thus, the second valve 162 and the third valve 163 are arranged between the first reaction chamber 110 and the pump chamber 120 in the first microfluidic connection channel. The pump chamber 120 is fluidically connected to the mixing chamber 130 via a second microfluidic connection channel. The fourth valve 164 is arranged in the second microfluidic connection channel. The pump chamber 120 is fluidically arranged between the first reaction chamber 110 and the mixing chamber 130.

[0029] The mixing chamber 130 is fluidically connected to the pump chamber 120 via the second microfluidic connecting channel. The fourth valve 164 is arranged between the pump chamber 120 and the mixing chamber 130 in the second microfluidic connecting channel. Thus, the mixing chamber 130 is fluidically connected to the first reaction chamber 110 via the second microfluidic connecting channel, the pump chamber 120, and the first microfluidic connecting channel. The first reaction product can be fed from the first reaction chamber 110 via the pump chamber 120 into the mixing chamber 130. A dilution medium can be or is stored upstream in the mixing chamber 130. The mixing chamber 130 is designed to mix the first reaction product from the first reaction chamber 110 with the dilution medium to create a diluted mixture.

[0030] The ventilation opening 140 is formed in the mixing chamber 130. Even if it is Fig. 1 due to the nature of the illustration, the ventilation opening 140 is arranged as a through-opening between the mixing chamber 130 and an outer surface of the microfluidic device 100. The ventilation opening 140 is formed as a compensating opening relative to an ambient pressure.

[0031] Between the second valve 162 and the third valve 163, according to the Fig. In the exemplary embodiment of the present invention shown in Figure 1, a branching point is arranged in the first microfluidic connecting channel between the first reaction chamber 110 and the pump chamber 120. At the branching point, a third microfluidic connecting channel branches off from the first microfluidic connecting channel. The third microfluidic connecting channel extends between the branching point and, for example, the four second reaction chambers 150. The third microfluidic connecting channel branches into four subchannels.

[0032] Each of the exemplary four second reaction chambers 150 is arranged between a fifth valve 165 and a sixth valve 166. Thus, according to the Fig. In the exemplary embodiment of the present invention shown in Figure 1, four fifth valves 165 and four sixth valves 166 are provided. Each of the fifth valves 165 is arranged in one of the sub-channels of the third microfluidic connecting channel. The second reaction chambers 150 are fluidically connected to the mixing chamber 130 via the microfluidic connecting channels. The diluted mixture from the mixing chamber 130 can be fed into the second reaction chambers 150 via the third microfluidic connecting channel. The second reaction chambers 150 are designed to perform a second polymerase chain reaction on the diluted mixture for analyzing the sample. The exemplary four sixth valves 166 are arranged in four sub-channels that combine in a microfluidic discharge channel.

[0033] Even if it is in Fig. 1 is not explicitly recognizable, then according to the Fig. In the embodiment of the present invention shown in Figure 1, at least one substance for carrying out the first polymerase chain reaction is stored upstream in the first reaction chamber 110. Furthermore, at least one first substance for carrying out the second polymerase chain reaction is stored upstream in the mixing chamber 130. Finally, at least one second substance for carrying out the second polymerase chain reaction is stored upstream in the second reaction chambers 150. The microfluidic device 100 is particularly designed to generate a diluted mixture with a mixing ratio that is suitable for at least partially excluding primers from the first polymerase chain reaction from the second polymerase chain reaction. The valves 161, 162, 163, 164, 165, and 166 can have interfaces that are designed to receive control signals.

[0034] According to one embodiment, a volume of the first reaction chamber 110 and a volume of the mixing chamber 130 are configured to effect a mixing ratio in the diluted mixture that is suitable for at least partially excluding primers from the first polymerase chain reaction from the second polymerase chain reaction in the second reaction chambers 150.

[0035] In other words, Fig. 1 shows a schematic top view of the microfluidic device 100 according to an embodiment. The microfluidic device 100 is designed, for example, as a microfluidic chip and comprises, among other things, the first reaction chamber 110 for carrying out a first polymerase chain reaction (PCR) as a multiplex PCR, the pumping chamber 120, the mixing chamber 130, and the second reaction chambers 150 for carrying out the second PCR as a singleplex polymerase chain reaction. Substances or reaction components for the first PCR, such as polymerase, different primer pairs for a multiplexed PCR, nucleoside triphosphates, in particular deoxyribonucleoside triphosphates or dNTP, PCR buffer, additives, and the like, are stored upstream in the first reaction chamber 110.Reaction components for the second PCR, such as dNTP, PCR buffer, and additives, are stored in the dilution medium in the mixing chamber 130. Polymerase, primer pairs, and, for example, real-time probes or probes for performing a real-time PCR, are stored in the second reaction chambers 150.

[0036] During operation of the microfluidic device 100, the sample or DNA sample is poured into the first reaction chamber 110 and mixed with the reaction components stored therein. When the first valve 161 and the second valve 162 are closed, reaction conditions for the first PCR are created. Subsequently, by means of a conveying device having the pump chamber 120 and the third valve 163 as well as the fourth valve 164 as pump valves, the first reaction product from the first PCR or the liquid volume of the first reaction chamber 110 is transferred into the mixing chamber 130 and mixed with the dilution medium located therein. Excess air from the mixing chamber 130 is displaced via the ventilation opening 140. The diluted mixture is distributed to the second reaction chambers 150 by means of the pump chamber 120 with the second valve 162 closed.Subsequently, the fifth valve 165 and sixth valve 166 are closed, creating reaction conditions for the second PCR. The reaction can be monitored either in real time, whereby quantitative information about an initial amount of each DNA molecule can be obtained, or measured at the end of the second reaction, allowing yes / no statements. According to the method described in . Fig. In the embodiment of the present invention shown in Figure 1, the mixing ratio or a dilution stage is determined by an amount of upstream dilution medium in the mixing chamber 130.

[0037] According to a related to the Fig. 1, the simplified embodiment of a microfluidic device 100 comprises, in addition to the first reaction chamber 110 and the mixing chamber 130, at least one second reaction chamber 150. A channel leading into the second reaction chamber 150 can, as in Fig. 1, branch off from a connecting channel between the first reaction chamber 110 and the mixing chamber 130, or alternatively lead from the mixing chamber 130 into the second reaction chamber 150. An optional pumping chamber 120 can be arranged at a suitable position in a connecting channel network connecting the chambers 110, 130, 150.

[0038] Fig. Figure 2 shows a schematic representation of a microfluidic device 100 for analyzing a sample of biological material according to an embodiment of the present invention. The microfluidic device 100 corresponds to the microfluidic device of Fig. 1 except that the microfluidic device 100 in Fig. 2 additionally has a seventh valve 267 and a storage chamber 270. Thus, the microfluidic device 100 in Fig. 2 shows the first reaction chamber 110, the pumping chamber 120, the mixing chamber 130, the ventilation opening 140, the four second reaction chambers 150, the valves 161, 162, 163, 164, 165, 166 and 267 and the storage chamber 270, merely by way of example.

[0039] The storage chamber 270 is fluidically connected to the branching point in the first microfluidic connecting channel between the first reaction chamber 110 and the pumping chamber 120 via a fourth microfluidic connecting channel. The seventh valve 267 is arranged in the fourth microfluidic connecting channel. The storage chamber 270 is designed to store the dilution medium. The storage chamber 270 is fluidically connected to the mixing chamber 130 via the fourth microfluidic connecting channel. Thus, the first reaction product in the mixing chamber 130 can be diluted with the dilution medium from the storage chamber 270 in an adjustable mixing ratio.

[0040] In other words, Fig. 2 shows a structure of the microfluidic device 100 in which different dilution levels of the first reaction product for the second PCR can be set. According to the Fig. In the embodiment of the present invention shown in Figure 2, the dilution medium for the second PCR is stored in the storage chamber 270. A reaction volume of the first PCR or the first reaction product is transferred into the mixing chamber 130 by means of the pump chamber 120. By means of the pump chamber 120, a defined amount of the dilution medium is also supplied from the storage chamber 270 to the mixing chamber 130 and mixed there with the first reaction product. This diluted mixture is then, as shown in Fig. 1, distributed among the second reaction chambers 150, where they are temperature-cycled during the second PCR. Thus, different dilution levels of the first reaction product in the diluted mixture can be set without any design changes.

[0041] Fig. Figure 3 shows a comparative representation of analysis results according to an embodiment of the present invention. The analysis results were obtained by manual pipetting using Eppendorf tubes (= reaction vessels). The results demonstrate that the basic idea (inactivation of primers from a first PCR reaction by dilution) works.

[0042] In other words, Fig. 3 shows a comparative representation of a gel electrophoresis of analysis results from a "Diluted Nested PCR" according to embodiments of the present invention, performed in a laboratory PCR device. The first reaction product or a PCR product of a first PCR, a multiplex PCR, was diluted by different dilution factors and used as so-called template DNA for the second PCR, in the form of several singleplex PCRs. A primer pair for the PVL gene was used. Some of the columns A, B, C, D, E, F, G, and H in Fig. 3 represent the analysis results obtained based on different dilution factors.

[0043] Column A shows a PCR product of the first PCR or a first reaction product. Column B shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 50 and used as template DNA. Column C shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 500 and used as template DNA. Column D shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 5000 and used as template DNA. Column E shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 5000 and used as template DNA. Column F shows a second reaction product ora PCR product of the second PCR, for which the first reaction product was diluted by a factor of 500,000 and used as template DNA. Column G shows a negative control, and column H shows a DNA ladder, purely exemplary of the 50 bp plus type.

[0044] Fig. Figure 4 shows a comparative representation of analysis results according to an embodiment of the present invention. The analysis results were obtained by manual pipetting using Eppendorf tubes (= reaction vessels). The results demonstrate that the basic idea (inactivation of primers from a first PCR reaction by dilution) works.

[0045] In other words, Fig. 4 shows a comparative representation of a gel electrophoresis of analysis results from a "Diluted Nested PCR" according to embodiments of the present invention, performed in a laboratory PCR device. The first reaction product or a PCR product of a first PCR, a multiplex PCR, was diluted by different dilution factors and used as so-called template DNA for the second PCR, in the form of several singleplex PCRs. A primer pair for the SSC1 gene was used. Some of the columns A, B, C, D, E, F, G, and H in Fig. 4 represent the analysis results obtained based on different dilution factors.

[0046] Column A shows a PCR product of the first PCR or a first reaction product, and column B shows a DNA ladder, purely as an example of the 50 bp plus type. Column C shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 50 and used as template DNA. Column D shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 500 and used as template DNA. Column E shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 5000 and used as template DNA. Column F shows a second reaction product or a PCR product of the second PCR, for which the first reaction product was diluted by a factor of 50,000 and used as template DNA. Column G shows a second reaction product ora PCR product of the second PCR, for which the first reaction product was diluted by a factor of 500,000 and used as template DNA. Column H shows a negative control.

[0047] Fig. 5 shows a flow diagram of a method 500 for analyzing a sample of biological material according to an embodiment of the present invention. The method 500 can be carried out using a microfluidic device having a first reaction chamber, a mixing chamber fluidically connected to the first reaction chamber, and at least one second reaction chamber fluidically connected to the mixing chamber. In particular, the method 500 can be carried out using a microfluidic device, as described, for example, with reference to Fig. 1 or Fig. 2 is described.

[0048] The method 500 includes a step 510 of performing a first polymerase chain reaction on the sample in the first reaction chamber to generate a first reaction product. The method 500 also includes a step 520 of mixing the first reaction product with a dilution medium in the mixing chamber to generate a diluted mixture. Furthermore, the method 500 includes a step 530 of performing a second polymerase chain reaction on the diluted mixture in the at least one second reaction chamber to analyze the sample.

[0049] According to one embodiment, in step 520 of mixing, control signals are generated for controlling at least one valve and additionally or alternatively at least one conveying device of the microfluidic device in order to effect a mixing ratio in the diluted mixture that is suitable for at least partially excluding primers from the first polymerase chain reaction from the second polymerase chain reaction.

[0050] In the following, embodiments of the present invention are described with reference to the Fig. 1 to 5 summarized and explained in other words.

[0051] The comparison images from the Fig. 3 and Fig. 4 show that when the first reaction product or the PCR product of the first PCR, the multiplex PCR, is diluted by a factor of 500 with dilution medium in the mixing chamber 130, a single, specific PCR product is generated in the at least one downstream singleplex PCR, the second PCR. Dilution of the first reaction product can prevent the primers of the first PCR from remaining active in the second PCR. Thus, only the desired singleplex PCR product can be generated in the second PCR, and not all other PCR products addressed by the multiplex primer pairs. In this way, increased reaction efficiency can be achieved with more economical use of reaction components and increased enzyme activity, which is why, for example, even small amounts of DNA sequences can be detected.

[0052] According to one embodiment, the dilution is carried out by a factor of 50. This can achieve a partial inactivation of the primer pairs of the first PCR, as described in Fig. 3, column B or Fig. 4, column C. The necessary specificity can be achieved by using real-time probes in the second PCR, which only provide signals in the presence of a specific DNA sequence. For example, this is achieved using the microfluidic device 100 of Fig. 2, for example, the first reaction chamber 110 has a volume of 10 microliters and the storage chamber 270, which contains the dilution medium with reaction components, has a volume of 490 microliters.

[0053] According to embodiments of the present invention, a thickness of the microfluidic device 100 may, for example, be 0.5 to 5 millimeters, wherein channel diameters of microfluidic channels may be 10 micrometers to 3 millimeters and volumes of chambers may be between 1 cubic millimeter and 1000 cubic millimeters.

[0054] An exemplary process flow for a first PCR comprises a parameter space with a number of 1 to 20 thermal cycles, whereby temperature cycles of 93 degrees Celsius to 96 degrees Celsius are provided for denaturation, 72 degrees Celsius for extension, and 54 degrees Celsius to 65 degrees Celsius for primer binding. It is also possible to omit the extension temperature step and thus perform a two-step PCR. For a second PCR, a parameter space comprises a number of 20 to 60 thermal cycles, whereby temperature cycles of 93 degrees Celsius to 96 degrees Celsius are provided for denaturation, 72 degrees Celsius for extension, and 54 degrees Celsius to 65 degrees Celsius for primer binding. It is also possible to omit the extension temperature step and thus perform a two-step PCR.For example, the number of polymerase chain reactions for the first PCR is between 1 and 20 reactions and for the second PCR between 1 and 50 reactions.

[0055] Measuring the success of a PCR reaction involves, for example, the use of hydrolysis probes, such as TaqMan probes, LightCycler probes, LoopTag probes, Scorpion primers, or molecular beacons. The reaction success can be determined by an increasing fluorescence signal. Other possible methods include electrical readout using ISFETs, impedance measurement, measurement of a surface-bound PCR product using interdigital electrode structures, or similar methods.

[0056] The exemplary embodiments described and shown in the figures are selected only as examples. Different exemplary embodiments can be combined with each other in their entirety or with regard to individual features. Furthermore, one exemplary embodiment can be supplemented by features of another exemplary embodiment.

[0057] Furthermore, the process steps presented here can be repeated and carried out in a different order than that described.

[0058] If an embodiment comprises an “and / or” link between a first feature and a second feature, this is to be read as meaning that the embodiment according to one embodiment has both the first feature and the second feature and according to another embodiment has either only the first feature or only the second feature.

Claims

[1] Microfluidic device (100) for analyzing a sample of biological material, the microfluidic device (100) having the following features: a first reaction chamber (110) configured to perform a first polymerase chain reaction on the sample to produce a first reaction product; a mixing chamber (130) fluidly connected or connectable to the first reaction chamber (110) and configured to mix the first reaction product with a dilution medium to produce a diluted mixture; and at least one second reaction chamber (150) which is fluidically connected or connectable to the mixing chamber (130) and is designed to carry out a second polymerase chain reaction on the diluted mixture in order to analyze the sample. [2] Microfluidic device (100) according to claim 1, wherein at least one substance for carrying out the first polymerase chain reaction is or can be stored upstream in the first reaction chamber (110), wherein the dilution medium is stored upstream in the mixing chamber (130), wherein at least one substance for carrying out the second polymerase chain reaction is stored upstream in the at least one second reaction chamber (150) and / or in the mixing chamber (130). [3] Microfluidic device (100) according to claim 1, comprising a storage chamber (270) which is fluidically connected or connectable to the mixing chamber (130) for storing dilution medium, wherein at least one substance for carrying out the first polymerase chain reaction is or can be stored upstream in the first reaction chamber (110), wherein the dilution medium is stored upstream in the storage chamber (270), wherein at least one substance for carrying out the second polymerase chain reaction is stored upstream in the at least one second reaction chamber (150), in the storage chamber (270) and / or in the mixing chamber (130). [4] Microfluidic device (100) according to one of the preceding claims, wherein a volume of the first reaction chamber (110) and a volume of the mixing chamber (130) are designed to effect a mixing ratio in the diluted mixture that is suitable for excluding primers from the first polymerase chain reaction from the second polymerase chain reaction. [5] Microfluidic device (100) according to one of the preceding claims, comprising a vent opening (140) formed in the mixing chamber (130) as a compensation opening relative to an ambient pressure. [6] Microfluidic device (100) according to one of the preceding claims, with a pump chamber (120) fluidically connected between the first reaction chamber (110) and the mixing chamber (130) and a plurality of valves (161, 162, 163, 164, 165, 166, 167), wherein a plurality of second reaction chambers (150) are provided, wherein the first reaction chamber (110) is arranged between a first pair of valves (161, 162), the pump chamber (120) is arranged between a second pair of valves (163, 164) and the plurality of second reaction chambers are arranged between a plurality of third pairs of valves (165, 166). [7] A method (500) for analyzing a sample of biological material, wherein the method is executable using a microfluidic device (100) having a first reaction chamber (110), a mixing chamber (130) fluidically connected to the first reaction chamber (110), and at least one second reaction chamber (150) fluidically connected to the mixing chamber (130), the method (500) comprising the following steps: performing (510) a first polymerase chain reaction on the sample in the first reaction chamber (110) to produce a first reaction product; Mixing (520) the first reaction product with a dilution medium in the mixing chamber (130) to produce a diluted mixture; and Performing (530) a second polymerase chain reaction on the diluted mixture in the at least one second reaction chamber (150) to analyze the sample. [8] Method (500) according to claim 7, wherein in the step (520) of mixing, control signals for controlling at least one valve (161, 162, 163, 164, 165, 166, 267) and / or at least one conveying device of the microfluidic device (100) are generated in order to effect a mixing ratio in the diluted mixture which is suitable for excluding primers from the first polymerase chain reaction from the second polymerase chain reaction. [9] Device designed to carry out all steps of a method (500) according to one of claims 7 to 8. [10] Computer program configured to perform all steps of a method (500) according to any one of claims 7 to 8. [11] A machine-readable storage medium having stored thereon a computer program according to claim 10.

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