WO2015162060A1

WO2015162060A1 - Microfluidic device and method for analysing a sample of biological material

Info

Publication number: WO2015162060A1
Application number: PCT/EP2015/058354
Authority: WO
Inventors: Jochen Rupp; Jochen Hoffmann
Original assignee: Robert Bosch Gmbh
Priority date: 2014-04-25
Filing date: 2015-04-17
Publication date: 2015-10-29
Also published as: DE102014221616A1

Abstract

The invention relates to a microfluidic device (100) for analysing a sample of biological material. The microfluidic device (100) has an amplification section (110) having at least one reaction chamber (111, 112, 113) for conducting a polymerase chain reaction having a multitude of cycles for an amplification of the sample. In this device, the at least one reaction chamber (111, 112, 113) is formed in order to enable denaturing of target molecules of the sample, primer hybridization of denatured target molecules of the sample, and elongation of primer-hybridized target molecules of the sample. The microfluidic device (100) also has at least one detection chamber (130) for repeated execution of detection reactions on denatured target molecules of the sample from different cycles of the polymerase chain reaction. In this case, the at least one detection chamber (130) is connected for fluidic purposes to the amplification section (110). In addition, the microfluidic apparatus (100) has at least one device for transport (120) for repeated conveying of the sample between the amplification section (110) and the at least one detection chamber (130).

Description

description

title

Microfluidic device and method for analyzing a sample of biological material

State of the art

The present invention relates to a microfluidic device for analyzing a sample of biological material, to a method for

Analyzing a sample of biological material, on a device which is designed to perform and / or to control all steps of such a method, to a corresponding computer program and to a corresponding storage medium. Usually, in microfluidic diagnostic systems, such. B. Lab on

Chip systems or LOC systems, a sample to be examined first amplified by means of polymerase chain reaction (PCR, polymerase chain reaction) and then analyzed on a microarray. The

DE 10 2009 035 270 A1 describes a one-way multiplex polymerase chain reaction chip and a polymerase chain reaction device.

Disclosure of the invention

Against this background, with the approach presented here, a microfluidic device for analyzing a sample of biological material, a method for analyzing a sample of biological material, a

Device that is designed to perform and / or control all steps of such a method, a corresponding computer program and a corresponding storage medium according to the main claims presented. Advantageous embodiments emerge from the respective subclaims and the following description.

According to embodiments of the invention, in particular a system can be provided in which amplification reactions, in particular a polymerase chain reaction (PCR), and detection reactions, in particular in a so-called microarray or MArray, can be linked in one component for sample analysis. Thus, for example, a spatial, thermal and fluidic separation of the two individual functions can be achieved in order to enable advantageous processing. In particular, a microfluidic system with integrated chambers and fluidic connections for carrying out a PCR and a nucleic acid microarray, which is likewise arranged in the microfluidic system, can be provided. By repeatedly conveying or reciprocating a reaction volume of a sample between chambers, which may be at different temperatures, for example, a PCR can be carried out. During this PCR, the reaction volume can be brought into contact with the DNA microarray. In this case, for example, immobilized oligonucleotides or

Probe microarrays interact with strands of a PCR product and fix the same.

Advantageously, according to embodiments of the invention, an improved array PCR can be made possible. For example, a miniaturized and integrated implementation of complex fluidic workflows for the specific detection of various molecules by means of structures and

Method according to embodiments of the invention in a faster process flow and with a simplified process management are made possible. In particular, a shortening of a total duration of a detection of genetic features can be achieved since, according to embodiments of the invention, PCR products can already be defined in a spatially resolved manner during the PCR

Time points can be detected. A detection reaction or

Read reaction or hybridization, with or without washing of the microarray, can in this case in particular at different times parallel to or

during the otherwise upstream PCR done. This allows real-time capability to be achieved. It results advantageously a maximum temporal resolution up to a single PC R cycle. The microfluidic device can also be self-contained during the process, for example, so that no sample volume is removed from the reactions during read-out, which can maximize the sensitivity of the overall system. A fluidic separation of amplification section and detection chamber or readout structure, for example, can enable independent fluidic processing of both functional areas, eg. B. Simultaneous washing of the array on continuation of an amplification reaction. A spatial separation of amplification section and detection chamber or readout structure

for example, can provide a simple optical access to the signals of the microarray, since no heating elements or the like

Amplification section obstruct an optical path to the microarray.

A microfluidic device for analyzing a sample of biological material is presented, wherein the microfluidic device has the following features: an amplification section with at least one reaction chamber for

Performing a polymerase chain reaction having a plurality of cycles for amplification of the sample, the at least one reaction chamber being configured to permit denaturation of target molecules of the sample, primer hybridization of denatured target molecules of the sample, and elongation of primer-hybridized target molecules of the sample; at least one detection chamber for repeated execution of

Detection reactions on denatured target molecules of the sample

different cycles of the polymerase chain reaction, wherein the at least one detection chamber is fluidically connected to the amplification section; and at least one transporting / conveying device for repeatedly conveying the sample between the amplifying section and the at least one detection chamber. The microfluidic device may comprise or be part of an analytical system, in particular a microfluidic lab-on-chip system or a chiplabor system for medical diagnostics, microbiological diagnostics or environmental analysis. The microfluidic device may be a chip, in particular with multiple layers, of a polymer material. As the sample, a liquid to be analyzed, typically a liquid or liquefied patient sample, e.g. Blood, urine, stool, sputum, cerebrospinal fluid, lavage, a flushed swab or liquefied tissue sample, or a sample of a non-human material. For example, cells contained in the sample may contain human cells, e.g. As blood cells or the like, animal cells or pathogenic cells or pathogens, eg. As viruses or microorganisms, such as. As bacteria or fungi, comprising DNA and / or RNA, ie nucleic acid molecules, as target molecules. The amplification section and the at least one detection chamber may be arranged spatially and additionally or alternatively fluidly separated from one another in the microfluidic device. The microfluidic device may comprise fluid lines, by means of which the at least one

Reaction chamber and the at least one detection chamber fluidly

connected to each other. Valves may be arranged in the fluid lines. In particular, for a correct analysis procedure, cells of a sample can be lysed before the PCR and subsequently the DNA can be purified.

According to one embodiment, the at least one device for the

Transport in the at least one reaction chamber and additionally or alternatively in the at least one detection chamber arranged, deflectable

Have membranes for displacing a volume of the sample. Such an embodiment offers the advantage that liquid volumes can be defined in a microfluidic channel connected to the respective chamber or in another chamber by direct volume displacement and can be easily relocated.

Also, the at least one device for transport with the

Amplification section and the at least one detection chamber fluidly connected pumping chamber. Such an embodiment offers the advantage that by means of the pumping chamber a discrete sample volume is particularly simply and effectively repeatedly between the amplification and the at least one detection chamber back and forth can be transferred.

Furthermore, tempering for tempering the at least one

Reaction chamber and the at least one detection chamber on respective

Target temperatures can be provided. In this case, the tempering means may be configured to temper the at least one reaction chamber to a first temperature and to temper the at least one detection chamber to a second temperature, wherein the first temperature and the second temperature may have different temperature values. The

Temperature control can have at least one temperature control. The temperature control means may have an associated tempering device for each chamber to be tempered. The temperature control means may be arranged in or on the microfluidic device. Alternatively, the

Temperature control means may be arranged separately from the microfluidic device and thermally coupled to the microfluidic device. Such an embodiment offers the advantage that chambers to be tempered can be brought to respective setpoint temperatures for carrying out respective reactions or held in the region thereof. Supported by a spatial separation of the functional areas amplification and detection respectively

Auslese can be made possible here a provision of separate heating areas for individual thermal requirements of amplification reactions and detection reactions or readout reactions. According to one embodiment, the amplification section and the at least one detection chamber can be fluidically connected to one another in a microfluidic network so that a cyclical and / or unidirectional sequence is made possible. The microfluidic network can be closed in this case, wherein an inlet and an outlet can be provided fluidly shut off. In this case, the amplification section and the at least one

Detection chamber be connected to each other via two fluidic paths. Such an embodiment offers the advantage that a repeated transfer of a sample volume between the amplification section and the at least one detection chamber can be further simplified. It can be a

Sample volume can be easily circulated. In this case, a bypass line connected in parallel with respect to the at least one detection chamber and additionally or alternatively a compensation chamber fluidically connected to the amplification section and the at least one detection chamber may be provided. The compensation chamber may be configured to represent or provide a compensation volume, a fluidic capacity or a throttle function. Such

Embodiment offers the advantage that the microfluidic device can also meet different or changing requirements of sample analyzes.

Also, the amplification section may have a plurality of fluidically interconnected reaction chambers. This can be a first

Reaction chamber may be formed to allow the denaturation of target molecules of the sample. In this case, a second reaction chamber may be formed to allow the primer hybridization of denatured target molecules of the sample. Furthermore, a third reaction chamber may be formed to allow the elongation of primary hybridized target molecules of the sample. In this case, the at least one device for transport for repeatedly conveying the sample between the reaction chambers may be formed. Thus, the sample or a sample volume can be located both between the reaction chambers and between the amplification section and the at least one

Detection chamber be promoted. The reaction chambers may be fluidly connected in series in the amplification section. Such

Embodiment offers the advantage that the sample processing can be further accelerated because, inter alia, each reaction chamber is individually preheated.

Furthermore, a method for analyzing a sample of biological material is presented, the method comprising the following steps:

Performing a denaturation of target molecules of the sample as a part of a polymerase chain reaction in an amplification section of a microfluidic device with at least one reaction chamber for Conducting a polymerase chain reaction with a plurality of cycles for amplification of the sample;

Conveying the sample from the amplification section into at least one detection chamber fluidically connected to the amplification section;

Performing detection reactions on denatured target molecules of the sample in the at least one detection chamber;

Returning the sample from the at least one detection chamber into the amplification section; and

Effecting primer hybridization of denatured target molecules of the sample and elongation of primer-hybridized target molecules of the sample in the amplification section.

The method may be advantageously carried out in conjunction with an embodiment of the aforementioned microfluidic device to analyze a sample of biological material.

In one embodiment, the steps may be repeated for each cycle of the polymerase chain reaction. Such an embodiment offers the advantage that a detection reaction can be performed at different times in parallel to or during the PCR, thus real-time capability can be achieved with a maximum temporal resolution of up to a single PCR cycle.

The approach presented here also provides a device which is designed to implement the steps of a variant of a method presented here

to implement, control or implement appropriate facilities. Also by this embodiment of the invention in the form of a device, the object underlying the invention can be solved quickly and efficiently. In the present case, a device can be understood as meaning an electrical device which processes sensor signals and outputs control and / or data signals in dependence thereon. The device may have an interface, which may be formed in hardware and / or software. In the case of a hardware-based embodiment, the interfaces can be part of a so-called system ASIC, for example, which contains a wide variety of functions of the device. However, it is also possible that the interfaces are their own integrated circuits or at least partially consist of discrete components. In a software training, the interfaces may be software modules that are present, for example, on a microcontroller in addition to other software modules.

An advantage is also a computer program product with program code, which on a machine-readable carrier such as a semiconductor memory, a

Can be stored in hard disk space 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,

especially when the program product is running on a computer or device.

The approach presented here will be described below with reference to the attached

Illustrated drawings by way of example. Show it:

Figures 1 to 4 are schematic representations of a microfluidic device with fluid conveying directions according to embodiments of the present invention;

5 is a flowchart of a method for analyzing a sample of biological material according to an embodiment of the present invention;

Fig. 6 is a schematic representation of a molecular staining process according to an embodiment of the present invention; and Fig. 7 is a diagram showing intensity signals for various amplified and hybridized DNA segments.

In the following description of favorable embodiments of the present invention are for the in the various figures

represented and similar elements acting the same or similar

Reference numeral used, wherein a repeated description of these elements is omitted. Fig. 1 shows a schematic representation of a microfluidic device

100 for analyzing a sample of biological material according to a

Embodiment of the present invention. Here, by way of example, an amplification section 110, three reaction chambers 111, 112 and 113, a first connection channel 114, a second connection channel 115, a pumping chamber 120, one of the microfluidic device 100 are shown

Detection chamber 130 or array chamber, a compensation chamber 140, an ambient line 150, an inlet 160, an outlet 170, a third

Connecting passage 181, a fourth valve 182, a fifth valve 183, a sixth valve 184, a seventh valve 185, an eighth valve 186, a ninth valve 187, a first fluid conveying direction A and a second fluid conveying direction B. The microfluidic device 100 is For example, a so-called chip lab or lab-on-chip (LOC). The valves 183 and 185 are in the present embodiment in particular without loss of generality equivalent, d. H. always open and close at the same time

The amplification section 110 here comprises the three reaction chambers 111, 112 and 113, which comprise a denaturation chamber 111, an elongation chamber 112 and a hybridization chamber 113, as well as the first valve or the first connection channel 114 and the second valve or the second connection channel 115. The first denaturation chamber 111 is fluidically connected to the elongation chamber 112 by means of a fluid line in which the first valve 114 is arranged. The elongation chamber 112 is fluidically connected to the other by means of a further fluid line, in which the second valve 115 is arranged

Hybridization chamber 113 connected. Reaction chambers 111, 112, and 113 are configured to undergo a polymerase chain reaction with a plurality of Perform cycles to amplify the sample. In this case, the denaturation chamber 111 is designed to perform or permit denaturation of target molecules of the sample. The

Hybridization chamber 113 is designed to perform primer hybridization of denatured target molecules of the sample. The

Elongationskammer 112 is formed to a elongation of

primerhybridisierten target molecules of the sample perform or to

enable. In addition, it should be noted that some of the valves are also designed as simple connection channels, that can not be switched to a closed state.

The detection chamber 130 is connected to the amplification section 110 fluidically or by means of fluid lines. According to the exemplary embodiment of the present invention illustrated in FIG. 1, the amplification section 110 and the detection chamber 130 are fluidically interconnected in a microfluidic circuit. In this case, the detection chamber 130 is fluidically between the denaturation chamber 111 and the

Hybridization chamber 113 connected. The detection chamber 130 has a microarray or nucleic acid microarray. The detection chamber 130 is designed to repeatedly perform detection reactions on denatured target molecules of the sample from different cycles of the polymerase chain reaction.

The pumping chamber 120 is fluidly connected to the amplification section 110 and the detection chamber 130. Specifically, the pumping chamber 120 is fluidly connected between the denaturation chamber 111 and the detection chamber 130. The pumping chamber 120 represents the device for transporting the microfluidic device 100. Although not explicitly shown in FIG. 1, the at least one device for transport in addition to the pumping chamber 120 may also be located in the reaction chambers 111, 112 and 113 and / or in the detection chamber 130 arranged deflectable membranes for displacing a volume of the sample. The pump chamber 120 or the device for transport is or are designed to repeatedly convey the sample between the amplification section 110 and the detection chamber 130. Also, the pumping chamber 120 is or is at least one Device for transport designed to repeatedly convey the sample between the reaction chambers 111, 112 and 113.

The compensation chamber 140 is fluidically connected between the detection chamber 130 and the amplification section 110. More precisely, that is

Compensation chamber 140 fluidly connected between the detection chamber 130 and the hybridization chamber 113. In this case, the compensation chamber 140 is fluidically connected to the detection chamber 130 and the amplification section 110 or the hybridization chamber 113. The compensation chamber 140 is designed to be a compensation volume, a fluidic capacity or a

To represent or provide throttle function.

The bypass line 150 is fluidly connected in parallel with respect to the detection chamber 130. The bypass line 150 extends from a first branch point located between the detection chamber 130 and the

Pumping chamber 120 is arranged, to a second branch point, which is arranged between the detection chamber 130 and the compensation chamber 140. The inlet 160 or fluid inlet of the microfluidic device 100 is located between the amplification section 110, more particularly the

Denaturation chamber 111, and the pumping chamber 120 arranged. The outlet 170 or fluid outlet of the microfluidic device 100 is in the

Bypass line 150 is arranged.

The third valve or the third connection channel 181 is between the

Hybridization chamber 113 and the compensation chamber 140 arranged. The fourth valve 182 is disposed between the equalizing chamber 140 and the second branching point. The fifth valve 183 is disposed between the second branching point and the detection chamber 130. The sixth

Valve 184 is disposed in the bypass 150 between the outlet 170 and the first branch point. The seventh valve 185 is disposed between the detection chamber 130 and the first branching point. The eighth valve 186 is disposed between the first branching point and the pumping chamber 120. The ninth valve 187 is between the pumping chamber 120 and the Inlet 160 arranged. The valves / connection channels 114, 115, 181, 182, 183, 184, 185, 186 and 187 are, for example, as diaphragm valves or

Membrane hoses executed in respective fluid lines. The microfluidic circuit or a fluidic circuit or sequence within the microfluidic device 100 has the inlet 160, the denaturation chamber 111, the first valve according to the embodiment of the present invention shown in FIG. 1 as well as with reference to the illustration in FIG 114, the elongation chamber 112, the second valve 115, the hybridization chamber 113, the third valve 181, the

Compensation chamber 140, the fourth valve 182, the second branch point, followed in a first branch, the fifth valve 183, the detection chamber 130 and the seventh valve 185 and in a second branch, which is fluidly parallel to the first branch, the bypass line 150th with the outlet 170 and the sixth valve 184, the first branch point, the eighth valve 186, the pumping chamber 120, and the ninth valve 187 follow.

The first fluid conveying direction A in this case runs clockwise from the

Denaturation chamber 111 to the pumping chamber 120. The second

Fluid conveying direction B runs counterclockwise from the

Pumping chamber 120 to the denaturation 111. On the

Fluid conveying directions A and B will be discussed in more detail below.

In other words, FIG. 1 shows a fluidic structure of the FIG

Microfluidic device 100 or a chip for combined

Performing an amplification reaction, d. H. a PCR, and a

Detection reaction, the latter by means of a microarray. Among other things, the structure comprises the three reaction chambers 111, 112 and 113, in which, with alternate fluid or liquid transport of a liquid

Sample volume between the reaction chambers 111, 112 and 113 a desired biochemical reaction, an amplification reaction, proceeds. The sample is examined for specific genetic characteristics. Furthermore, the microfluidic device 100 here comprises the pumping chamber 120 and a fluidic path which includes the detection chamber 130 or array chamber with a nucleic acid microarray therein. During the biochemical reaction or amplification reaction, a reaction volume by means of the pumping chamber 120 in the detection chamber 130 can be introduced to determine a course of the reaction and a composition of the sample, so perform a detection reaction. The microfluidic device 100 has two fluidic paths to the areas of amplification and

Be able to process the detection reaction individually fluidically.

The reaction chambers 111, 112 and 113 and the detection chamber 130 can each have, as a device for transport, an elastic membrane which can be deflected into the respective chamber. By such a direct

Volume displacement can be liquid volumes in a connected to the respective chamber microfluidic channel and / or relocate to another chamber. Although it is likewise not explicitly shown in FIG. 1, different regions of the microfluidic device 100, in particular the reaction chambers 111, 112 and 113 and the detection chamber 130, are through

Temperature control to temperature target temperatures. In other words, the reaction chambers 111, 112 and 113 and the detection chamber 130, for example, by heaters, z. B. in the microfluidic device 100 integrated or externally applied heater to be locally tempered. As a result, the respective regions can be exposed to different temperatures, which are required, for example, by a biochemical detection reaction or polymerase chain reaction (PCR). A combined amplification and detection reaction is defined in particular by fluidic, thermal and biochemical protocols.

For example, in operation of the microfluidic device 100, a sample solution in a PCR mix in the denaturation chamber 111 becomes

Provided. The area of the denaturation chamber 111 is tempered for a denaturation step, for example to temperatures between 90 degrees Celsius and 98 degrees Celsius. The sample solution is typically allowed to linger in the denaturation chamber 111 for between 2 and 60 seconds. In a further step, the sample volume in the

Hybridization chamber 113 transferred. The area of the hybridization chamber 113 is tempered, for example, to temperatures between 45 degrees Celsius and 72 degrees Celsius. The sample solution is for this so-called Annealing step typically between 2 and 60 seconds in the

Hybridization chamber 113 allowed to rest. In a further step, the sample volume is transferred into the elongation chamber 112. The region of the elongation chamber 112 is tempered, for example, to temperatures between 68 degrees Celsius and 76 degrees Celsius. The sample solution is allowed to remain in the elongation chamber 112 for typically between 10 and 120 seconds for this so-called elongation step. These steps are usually carried out cyclically for the polymerase chain reaction, for example, between 10 and 40 times. After amplification of DNA sections as just described, the reaction volume is cyclically brought into contact with the microarray in the detection chamber 130.

For detection of amplified DNA sections, the sample is introduced into the detection chamber 130 after a denaturation step carried out in the denaturation chamber 111. This will allow that

molten PCR product strands interact with probes of the microarray disposed in the detection chamber 130, and in the case of

Complementarity of nucleic acid sequences can be fixed by the probes. For example, these steps are cycled between 1 and 40 times.

According to the embodiment of the present invention shown in Fig. 1, the sample volume after 10 to 40 cycles of the

Amplification reaction after denaturing or melting in the denaturation chamber 111 along the first fluid conveying direction A via the reaction chambers 112 and 113 promoted or introduced into the detection chamber 130. Thereafter, the sample volume along the second

Fluid conveying direction B via the same path in the opposite direction via the reaction chambers 113 and 112 back into the Denaturierungskammer 111 or promoted. The conveying of the volumes back and forth over the described path between the Denaturierungskammer 111 and the

Detection chamber 130 thus takes place, for example, between once and 40 times.

FIG. 2 shows a schematic representation of the microfluidic device 100 from FIG. 1 with a different sequence of the fluid conveying directions A and B. Thus The illustration in FIG. 2 also corresponds to the illustration from FIG. 1 with the exception of the fluid conveying directions A and B. According to FIG

Embodiment of the present invention, the sample volume after the 10 to 40 cycles of the amplification reaction after denaturation in the denaturation chamber 111 along the second

Fluid conveying direction B promoted or transferred into the pumping chamber 120 and conveyed from there into the detection chamber 130 or introduced. Thereafter, the sample volume along the first fluid conveying direction A via the same path via the pumping chamber 120 directly into the denaturation 111

promoted or led back. The conveying of the volumes back and forth over the described path between the Denaturierungskammer 111 and the

Detection chamber 130 thus takes place, for example, between once and 40 times.

FIG. 3 shows a schematic representation of the microfluidic device 100 from FIG. 1 or FIG. 2 with only the first fluid conveying direction A. Thus, the representation in FIG. 3 also corresponds to the illustration from FIG. 1 or FIG. 2, with the exception of FIG only one, here the first fluid conveying direction A. According to the embodiment of the present invention shown in FIG. 3, the sample volume after the 10 to 40 cycles of the amplification reaction after denaturing in the denaturation chamber 111 along the first fluid conveying direction A via the reaction chambers 112 and 113 conveyed or introduced into the detection chamber 130. Thereafter, the sample volume is further conveyed or transferred along the first fluid conveying direction A by means of the pumping chamber 120 directly back into the denaturation chamber 111. Feeding the volumes back and forth over the described

The path between the denaturation chamber 111 and the detection chamber 130 thus occurs, for example, between once and 40 times.

4 shows a schematic representation of the microfluidic device 100 from FIG. 1, FIG. 2 or FIG. 3 with only the second fluid conveying direction B.

Thus, the representation in FIG. 4 also corresponds to the illustration from FIG. 1, FIG. 2 or FIG. 3, with the exception of only one, here the second one

Fluid Delivery Direction B. According to the embodiment of the present invention shown in Fig. 4, the sample volume after the 10 to 40 cycles of the amplification reaction after denaturing in the Denaturation chamber 111 along the second fluid conveying direction B in the pumping chamber 120 promoted or transferred and from there into the

Detection chamber 130 promoted or initiated. After that it will be

Sample volume continues along the second fluid conveying direction B via the reaction chambers 113 and 112 back into the denaturation 111 initiated or promoted. The conveying of the volumes back and forth over the described path between the Denaturierungskammer 111 and the

Detection chamber 130 thus takes place, for example, between once and 40 times.

With reference to FIGS. 1 to 4, in other words, further characteristics, aspects and / or features of

Embodiments of the present invention received.

It can via the fluidic path, the detection chamber 130 and

Microarray chamber, a washing solution are rinsed while the sample volume is in one of the reaction chambers 111, 112 and 113. As a result, substances which inhibit readout reaction, such as fluorophores, can be washed away.

Optionally, the rinse solution may be a PCR buffer, which may comprise, for example, all the components mentioned in the previous section. Preferably, the PCR buffer does not comprise primers. If such a rinse solution is rinsed into the detection chamber 130 and typically lingers there for between 1 and 60 seconds, the microarray probes will be in the

Extended primer extension reaction complementary to a hybridized to these probes PCR product strand. This increases one

Melting temperature and thus stability of a bonded to a surface of the microarray DNA duplex.

The microfluidic device 100 is formed, for example, from polymer substrates. In the polymer substrates, features of the microfluidic device 100 may be formed, for example, by milling, injection molding, hot stamping or laser structuring. The microarray of the detection chamber 130 can either be formed directly in the polymer or as an insert, For example, made of glass, be incorporated into the polymer layer structure.

Material examples include for such a polymer substrate in particular thermoplastics, for. PC, PP, PE, PMMA, COP, COC, PEEK or the like, for a polymer membrane usable for the valves 114, 115, 181, 182, 183, 184, 185, 186 and 187 and the device for transportation especially elastomer, thermoplastic elastomer, TPU, TPS, thermoplastics,

Hot-adhesive films, sealing films for microtiter plates, latex or the like.

An exemplary biomolecule formation comprises 5 to 100 nucleotide probe lengths, thiol group linker molecules,

Amino groups, gold, Glutharaldehyde, Acrydite ™ or the like, which are connected for example via a carbon chain with the probe, PCR primer having a length between 5 and 100 nucleotides, wherein a

Melting temperature of the two primers can differ greatly, hot-start polymerases, proof reading polymerases, etc. as polymerases and a diameter of a spot from 1 to 500 microns. Exemplary dimensions of the microfluidic device 100 include a

Thickness of a polymer substrate from 0.5 mm to 5 mm, a

Channel diameters in polymer substrates of 10 microns to 3 millimeters, a thickness of a polymer membrane of 5 to 500 microns and a volume of cavities in the polymer substrates of 1 cubic millimeter to 1000 cubic millimeters. Exemplary pressures for an actuation of a

Polymer membrane are between 0.2 bar and 2 bar.

FIG. 5 shows a flowchart of a method 500 for analyzing a sample of biological material according to an embodiment of the invention

present invention. The method 500 may be described in conjunction with

Use of a microfluidic device such as one of

microfluidic devices of Fig. 1 to Fig. 4 are advantageously carried out. Here, the method 500 includes a step 510 of performing a denaturation of target molecules of the sample as a substep of a polymerase chain reaction. The step 510 of performing is in one

Amplification section of a microfluidic device having at least one reaction chamber for carrying out a polymerase chain reaction with a

Plurality of cycles for amplification of the sample executable.

Then, the method 500 includes a step 520 of conveying the sample from the amplification section into at least one of fluidically

Amplification section connected detection chamber. Also, that shows

Method 500 includes a step 530 of performing detection reactions on denatured target molecules of the sample in the at least one detection chamber.

Thereafter, the method 500 includes a step 540 of returning the sample from the at least one detection chamber into the amplification section. Subsequently, the method 500 includes a step 550 of effecting primer hybridization of denatured target molecules of the returned sample and elongation of primed hybridized target molecules of the returned sample in the amplification section.

According to the embodiment of the present invention shown in FIG. 5, in the method 500, steps 510 to 550 are repeated or re-executed in each cycle of the polymerase chain reaction.

According to one embodiment, the method 500 further comprises a step 560 of completing a molecular staining process of

primer-hybridized target molecules. Step 560 of completing the molecular staining process will be described below in particular

Referring to Fig. 6 in more detail.

FIG. 6 shows a schematic representation of a molecular staining process 600 or staining principle of primer-hybridized target molecules according to an exemplary embodiment of the present invention. The molecular staining process 600 is, for example, in the step of completing according to the method Fig. 5 completed. In Fig. 6 are a first partial view A, a second

Partial representation B and a third partial view C of the staining process 600 shown. In the first partial view A, a product strand 601 and biotin

Desoxvuridine triphosphates 602 and biotin-dUTPs, respectively. For example, in addition to typical components such as salts, polymerase, PCR primers, nucleotides, and reaction enhancing agents, a PCR reaction mix also contains biotin-dUTPs 602. For staining process 600, these particular nucleotides, i.e. H. the biotin-dUTPs 602, during the reaction or PCR in the

Product strand 601 installed. In the second partial view B, a strand 604 connected to a probe of a microarray 603 with built-in biotin dUTPs 602 is shown. The third partial view C hereby corresponds to the second partial view B, with the exception that additionally streptavidin-conjugated fluorescence molecules 605 or streptavidin molecules are shown. In the third partial view C it is shown how the strand 604 connected to the probe of the microarray 603 is fluorescently stained. For this purpose, a rinsing or staining solution in a binding buffer contains the streptavidin-conjugated fluorescence molecules 605. The streptavidin-conjugated fluorescence molecules 605 bind to the biotin-dUTPs 602 or biotin molecules and thus mark biotin-containing PCR product bound to the probe of the microarray 603.

Advantageously, depending on the thymine content of a PCR product, an amount of dUTPs used in the PCR, a length of PCR

Products, etc. many fluorescence molecules 605 concentrate on a spot of the microarray 603, resulting in a strong fluorescence signal. This makes it possible to detect even small amounts of a PCR product with the microarray 603. This detection can also take place by means of a suitable microfluidic process, for example in real time.

According to one embodiment, a PCR reaction mix can only contain biotin-dUTPs, but not deoxythymidine triphosphates or dTTPs. Thus, in a synthesis of a product strand complementary to adenine bases exclusively biotinkonjugierte uracyl bases of a Install polymerase. For example, to minimize or eliminate interference with PCR efficiency, biotin-dUTPs and dTTPs are used at a ratio of five to one to one to five. Furthermore, in addition to biotin dUTPs and dTTPs, uridine triphosphates or UTPs can also be used in the ratio x: y: z, where x, y, z are the numbers between one and six in all

Combinations can take. Thus, generated PCR products by means of a UNG digestion (UNG = uracil DNA glycosylase) for a

Amplification be made unusable in another PCR. This is an advantageous biochemical measure to combat a series of DNA contaminations.

For staining, according to one embodiment, a reagent is used which contains sodium phosphate buffer in one

Concentration between 10 and 1000 millimoles, advantageously between 50 and 150 millimoles, with a pH between 6 and 9, advantageously 7.2. To minimize non-specific interactions with surfaces, 0.1% Tween 80 or Tween 20 may be added. Here, fluorescence-conjugated streptavidin 605 is added, for example, in concentrations of 0.1 to 100 micrograms per milliliter, advantageously from 1 to 10 micrograms per milliliter. A duration of staining can be between 0.1 and 30

Minutes, advantageously between 1 and 10 minutes.

The dyeing solution according to an embodiment of the

Hybridization be added directly or after an upstream washing step on the microarray 603. Advantage of direct addition is that one

Washing step can be saved and thus results in a saving of time and reagents. After staining is done either with a

Wash solution or washed successively with two washings. When using a washing solution, the following parameters can be used: 100 millimoles, sodium phosphate buffer, pH 7.2, between 0.1 and 10 minutes

Incubation, advantageously between 1 and 5 minutes. When using two wash solutions, the following parameters can be used: for the first wash solution 100 millimoles, sodium phosphate buffer, pH 7.2, between 0.1 and 10 minutes incubation, advantageously between 1 and 5 minutes; for the second wash 10 millimoles, sodium phosphate buffer, pH 7.2, between 0.1 and 10 minutes incubation, advantageously between 1 and 5 minutes. Thereafter, the microarray 603 is either blown dry or read out with a membrane pressed onto the surface. In other words, a method is provided in which the on the

Microarray 603 DNA sections attached after hybridization are molecularly stained. This allows the won

Amplify fluorescence signals and thus improve a sensitivity of the analysis, since it can be avoided, detection molecules, mostly

Fluorophores accumulate on a two-dimensional surface to provide well-detectable signals against the background. Thus, a microfluidic procedure with chemical reagents is presented to attach additional fluorophores to DNA sections attached to a microarray 603. The sensitivity of a microarray 603 can be increased without adapting the microarray, for example, to probe design, binding chemistry,

To make carrier material, readout principle. The sensitivity of microarrays 603 can be improved because a number of fluorophores per bound DNA segment can be increased, for example, more than doubled. By means of a suitable microfluidic process, it is also possible to enable signals to be amplified by molecular staining in subsections of the hybridization, thereby enabling read-out during the reaction. This is cost-effective, since in particular fluorescently labeled primers can be dispensed with. Polymer substrates may, for example, be patterned appropriately by milling, injection molding, hot stamping or laser structuring to allow implementation of the method. The microarray 603 can be either directly in

Polymer be formed or introduced as an insert, for example made of glass in the polymer layer structure.

Figure 7 shows a bar graph 700 with intensity signals for various amplified and hybridized DNA segments. The different amplified and hybridized DNA sections are plotted on an abscissa axis of the bar graph 700 and plotted on an ordinate axis of the column

Column chart 700 is a count in counts applied. In particular, signal amplification by staining with streptavidin-Cy3 D24 is also shown. For each DNA segment analyzed, three intensity signals or signals 710, 720, 730 are recorded. A first signal 710 represents a stained microarray, a second signal 720

represents a non-stained microarray and a third signal 730 represents a non-template control. The microarrays were thus either specifically stained or not after hybridization. It can be seen from FIG. 7 that significantly more first signals 710 are higher than a detection limit defined at 1000 counters than second signals 720.

The embodiments described and shown in the figures are chosen only by way of example. Different embodiments may be combined together or in relation to individual features. Also, an embodiment can be supplemented by features of another embodiment.

Furthermore, the method steps presented here can be repeated as well as executed in a sequence other than that described.

If an exemplary embodiment comprises an "and / or" link between a first feature and a second feature, then this is to be read so that the embodiment according to one embodiment, both the first feature and the second feature and according to another embodiment either only first feature or only the second feature.

Claims

claims

A microfluidic device (100) for analyzing a sample

biological material, the microfluidic device (100) comprising: an amplification section (110) having at least one

A reaction chamber (111, 112, 113) for conducting a polymerase chain reaction having a plurality of cycles for amplification of the sample, wherein the at least one reaction chamber (111, 112, 113) is adapted to denature target molecules of the sample, a primer hybridization allow denatured target molecules of the sample and elongation of primer-hybridized target molecules of the sample; at least one detection chamber (130) for the repeated execution of detection reactions on denatured target molecules of the sample from different cycles of the polymerase chain reaction, wherein the at least one detection chamber (130) fluidly with the

Amplification section (110) is connected; and at least one transporting device (120) for repeatedly conveying the sample between the amplification section (110) and the at least one detection chamber (130).

2. Microfluidic device (100) according to claim 1, characterized

characterized in that the device for transport in the at least one reaction chamber (111, 112, 113) and / or in the at least one detection chamber (130) arranged, deflectable membranes for displacing a volume of the sample. Microfluidic device (100) according to one of the preceding claims, characterized in that the device for transport have a pumping chamber (120) fluidically connected to the amplification section (110) and the at least one detection chamber (130).

Microfluidic device (100) according to one of the preceding claims, characterized by tempering means for controlling the temperature of the at least one reaction chamber (111, 112, 113) and the at least one detection chamber (130) to respective target temperatures.

Microfluidic device (100) according to one of the preceding claims, characterized in that the amplification section (110) and the at least one detection chamber (130) are fluidically interconnected in a microfluidic network, so that a cyclical and / or unidirectional sequence is made possible.

Microfluidic device (100) according to one of the preceding claims, characterized by a bypass line (150) connected in parallel with respect to the at least one detection chamber (130) and / or a compensation chamber fluidically connected to the amplification section (110) and the at least one detection chamber (130) ( 140).

Microfluidic device (100) according to one of the preceding claims, characterized in that the amplification section (110) has a plurality of fluidically interconnected

Reaction chambers (111, 112, 113), wherein a first

Reaction chamber (111) is formed to allow the denaturation of target molecules of the sample, wherein a second

Reaction chamber (113) is formed to allow the primer hybridization of denatured target molecules of the sample, wherein a third reaction chamber (112) is formed to allow the elongation of primer-hybridized target molecules of the sample, wherein the Device for transport (120) for repeatedly conveying the sample between the reaction chambers (111, 112, 113) are formed.

A method (500) of analyzing a sample of biological material, the method (500) comprising the steps of:

Performing (510) denaturing target molecules of the sample as a part of a polymerase chain reaction in one

An amplification section (110) of a microfluidic device (100) having at least one reaction chamber (111, 112, 113) for conducting a polymerase chain reaction having a plurality of cycles for amplification of the sample;

Conveying (520) the sample from the amplification section (110) into at least one detection chamber (130) fluidly connected to the amplification section (110);

Perform (530) detection reactions on denatured

Target molecules of the sample in the at least one detection chamber (130);

Feeding back (540) the sample from the at least one

Detection chamber (130) in the amplification section (110); and

Effecting (550) a primer hybridization of denatured

Target molecules of the returned sample and an elongation of primerhybridisierten target molecules of the returned sample in the amplification section (110).

A method (500) according to claim 8, characterized in that the steps (510, 520, 530, 540, 550) are repeated for each cycle of the polymerase chain reaction.

The method (500) of claim 8 or 9, characterized by a step (560) of performing a molecular staining process (600) of the primer hybridized target molecules.

11. Device which is designed to perform and / or control all the steps of a method (500) according to one of claims 8 to 10.

12. Computer program that is set up to take all the steps of a

Method (500) according to any one of claims 8 to 10 perform and / or to control.

13. A machine-readable storage medium with a computer program stored thereon according to claim 12.