EP3060678A1

EP3060678A1 - Analysis unit for carrying out a polymerase chain reaction, analysis device, method for operating such an analysis unit and method for producing such an analysis unit

Info

Publication number: EP3060678A1
Application number: EP14783585.4A
Authority: EP
Inventors: Franz Laermer; Juergen Steigert; Jochen Hoffmann
Original assignee: Robert Bosch GmbH
Current assignee: Robert Bosch GmbH
Priority date: 2013-10-23
Filing date: 2014-10-06
Publication date: 2016-08-31
Also published as: JP2016540501A; CN105637102A; WO2015058950A1; KR20160067872A; US20160251698A1; DE102013221525A1

Abstract

The invention relates to an analysis unit (100) for carrying out a polymerase chain reaction, wherein the analysis unit (100) comprises a lid element (130) with at least one lid recess (135) and a base element (150) with at least one base recess (152), wherein the base recess (152) is arranged opposite the lid recess (135) in order to form a reaction chamber (110). The analysis unit (100) further comprises a film (140) which is arranged, at least in the region of the lid recess (135), between the lid element (130) and the base element (150). The analysis unit (100) also comprises at least one channel (160a, 160b) which is formed between the lid element (130) and the base element (150) for channeling a fluid into and/or out of the base recess (152) of the reaction chamber (110). Finally, the analysis unit (100) comprises a probe carrier (155) which is arranged in the base recess (152) and which comprises at least one indicator material as the probe (157), for identifying a biochemical material, wherein the indicator material on the probe carrier (155) is in a solid aggregate state.

Description

description

title

Analvseeinheit for performing a polymerase chain reaction,

Analvsevorrichtung, method for operating such Analvseeinheit and methods of making such Analvseeinheit

State of the art

The present invention relates to an analysis device for

Carrying out a polymerase chain reaction, an analyzer

Method for operating an analysis unit, methods for producing an analysis unit and a corresponding computer program product.

Microfluidic diagnostic systems such as Lab-on-a-Chips (LOCs) allow the miniaturized and integrated execution of complex workflows for the specific detection of various molecules. In published LOC systems, the sample material to be examined is first amplified by means of PCR and then analyzed on a microarray (see, for example, J. Choi et al .: An integrated allele-specific polymerase chain reaction-microarray chip for multiplex single nucleotide polymorphism typing, Lab on a Chip, vol. 12, 2012). The combination of both operations and the multi-step process control require a longer process time and more complex processes

Litigation. Disclosure of the invention

Against this background, the approach presented here becomes one

Analysis device for carrying out a polymerase chain reaction, an analysis device, a method for operating an analysis unit, on

Method for producing an analysis unit and to a corresponding

Computer program product presented in accordance with the main claims. Advantageous embodiments emerge from the respective subclaims and the following description.

The approach presented here provides an analysis unit for carrying out a polymerase chain reaction, the analysis device having the following features:

a lid member having at least one lid recess;

a bottom member having at least one bottom recess, wherein the bottom recess of the lid recess is disposed opposite to form a reaction chamber;

a film which, at least in the region of the lid recess between the

Cover element and the bottom element is arranged;

at least one channel between the cover element and the

Floor element is adapted to a fluid in the bottom recess of the

Initiate and / or divert reaction chamber; and

a probe carrier arranged in the bottom recess, which probe at least one indicator material for identifying a biochemical

Material, wherein the indicator material on the probe carrier has a solid state of aggregation.

By a cover element and / or a bottom element can be understood, for example, a component which is made of a plastic, in particular of a polymer. By a recess, for example, a depression in the cover element or the bottom element can be understood. A film can be understood as meaning a flat, flexible element, such as a plastic layer. The film may be, for example, fluid-impermeable. Under a probe carrier, a rigid,

be understood in particular element on which the indicator material is arranged and fixed. By way of example, an indicator material can be understood as meaning a chemical reagent or a biomolecule which, on contact with a predetermined biochemical material, has a

Change of state undergoes which can be identified (for example, by optical analysis) and / or evaluated. By an identification on the one hand a recognition of the presence of a predetermined material itself and / or a recognition of a quantity and / or a quality of the

be understood predetermined material. The approach presented here is based on the finding that it is possible to carry out a solid-phase reaction in the area of biochemical analysis processes directly in a single reaction chamber in which a liquid-phase reaction of a biochemical reaction process also takes place. Mostly that

Indicator material as a solid phase, i. is in solid state, the entire biochemical reaction process can be carried out in a small and compact analysis unit. The starting material to be analyzed as well as any required catalyst materials, for example, for the achievement or synthesis of intermediates of the biochemical

Reaction process are required, can be introduced in this case in the form of a fluid through the channel in the reaction chamber or the Bodenausnehmung.

The approach presented here has the advantage that now a very simple technical possibility for carrying out a biochemical reaction is opened without intermediate products of the biochemical reaction

different process stages would have to be merged manually. In particular, it can be avoided that an intermediate product of a first partial reaction, in which a liquid-phase reaction is carried out, has to be moved manually into a separate compartment, by a second

Perform partial reaction in which a solid-phase reaction takes place. The approach presented here thus enables a much more cost-effective and faster execution of a desired biochemical reaction,

especially if this biochemical reaction has two separate parts or

Has partial reactions that are based on materials in different

Aggregate states based.

An embodiment of the present invention in which the film is fastened to the cover element is advantageous, in particular wherein the film fluid-tightly closes an opening of the lid recess opposite the through-opening and / or wherein the cover element has a passage opening in the region of the lid recess. Such an embodiment of the present invention offers the advantage that a fluid or another material in the bottom recess or the reaction chamber can be very easily (further) moved, for example, from the bottom recess or the editorial board and / or in another

Press reaction chamber. For example, this movement of the fluid or of the other material can take place by a pressure on the lid element in the region of the lid recess which, via the film, acts on the fluid in the lid

Floor recess is transmitted. Such a movement of the fluid or of the other material can be carried out particularly easily if the cover element has a passage opening in the region of the cover recess, via which a pneumatic pressure is introduced into the cover recess, and thus exerts a pressure on the film which forms

as a result bulges in the area of the bottom recess.

According to a particularly favorable embodiment of the present invention, the cover element may have at least one further cover recess and the bottom element at least one further bottom recess, wherein the further cover recess of the further bottom recess is arranged opposite and wherein the bottom recess and the other

Floor recess are fluidly coupled by means of the channel. Such an embodiment of the present invention offers the advantage that two different reaction chambers can be provided, so that, for example, different process stages of the biochemical reactions, which for example require different reaction temperatures, can also be carried out in different regions of the analysis unit (that is, the different ones of the reaction chambers). This advantageously allows a very flexible use of the analysis unit for different biochemical reactions.

In order to be able to demonstrate particularly flexible and precise individual biochemical materials, for example, for a reaction with a

Indicator material require different reaction temperatures, according to a further embodiment of the present invention, a further probe carrier may be arranged in the further bottom recess, which has as another probe at least one further indicator material for identifying another biochemical material, wherein the further indicator material on the other probe carrier a solid state having. According to a further embodiment of the present invention, the bottom recess can also separate the channel into at least two subchannels, wherein at least one subchannel enables a fluidic connection with an external environment of the analysis device, in particular wherein at least one subchannel has a valve. Such an embodiment of the present invention offers the advantage of a particularly simple and / or controllable filling or removal of material, in particular a fluidic material from the bottom recess or the reaction chamber. In particular, in order to be able to repeat several process stages cyclically, in a particularly favorable embodiment of the present invention, the sub-channels are fluidically coupled or coupled to each other at an end remote from the bottom recess, in particular wherein the sub-channels are components of an annular channel. Such an embodiment of the present invention has the advantage that a biochemical reagent, for example, in different Bodenausnehmungen or

Reaction chambers can be carried out by cyclic further displacement over the sub-channels, wherein a direction of movement for the reagent need not be changed.

An embodiment of the present invention is particularly favorable, in which the probe carrier as probe at least one additional one additional

Indicator material for identifying at least one additional biochemical material, wherein the additional indicator material on the

Probe carrier has a solid state of aggregation. Such

Embodiment of the present invention has the advantage that on a single probe carrier several different biochemical materials and / or different concentrations of such a material can be identified or detected particularly cost-effective, fast and technically easy to implement.

In order to ensure that an indicator material remains arranged on the probe carrier at a predefined position, the probe carrier can, according to a favorable embodiment of the present invention, have at least one probe carrier recess in which the at least one probe carrier has Indicator material is arranged, in particular wherein the

Sondenträgerausnehmung having a predetermined minimum depth.

Furthermore, according to one embodiment, the probe carrier can also be designed and arranged such that a reaction of a substance with the indicator material for identifying the biochemical material can be carried out during the polymerase chain reaction. Such an embodiment of the present invention offers the advantage of a particularly compact design of the analysis unit, wherein the analysis unit can also be produced very inexpensively.

Also, the approach presented here creates an analysis device for operating an analysis unit according to a variant presented here, wherein the

Analysis device has at least the following features:

a holder for placing and / or holding the analysis unit during operation of the analysis device;

a tempering unit for controlling the temperature of a fluid or a solid in the analysis unit held by the holder; and

an evaluation unit for evaluating a change in the

Indicator material.

For example, a holder may also be understood to mean a bearing surface on which the analysis unit is placed during the operation of the analysis device. A tempering unit may be understood to be a unit which heats or cools at least one section of the analysis unit.

An evaluation unit can be understood as meaning a unit which detects a change in state of the at least one indicator material, for example by optical analysis of electromagnetic radiation converted or emitted by the indicator material (or by an indicator material changed by the biochemical material). Such

Embodiment of the present invention offers the advantage of a very compact analysis of a biochemical material with an inexpensive and easy-to-provide analysis unit.

An embodiment of the present invention in which the temperature control unit is designed to be different at the same time is particularly favorable To bring sections of the analysis unit to a different temperature. Such an embodiment of the present invention offers the advantage that different partial reactions, which require different reaction temperatures, can be carried out simultaneously in different sections of the analysis unit. In this way, an analysis of a biochemical material can be performed very quickly with a technically simple to produce analysis unit.

Furthermore, the approach presented here provides a method for operating an analysis unit according to a variant presented here, the method having the following steps:

Providing the analysis unit;

Applying an analyte to the analyzing unit and / or catalytic material to perform a reaction in the analysis unit; and

Evaluating a change in the indicator material on the probe carrier.

Such an embodiment of the present invention offers the advantage of a particularly rapid and inexpensive way of analyzing a biochemical material.

Also favorable is an embodiment of the present invention in which the steps of loading and evaluating are carried out at least partially simultaneously. Such an embodiment of the present invention offers the advantage that, for example, different sub-steps of

Polymerase chain reaction can be carried out simultaneously or in parallel, so that on the one hand can be analyzed very quickly a result of the polymerase chain reaction and on the other hand, the implementation of the polymerase chain reaction technically very simple, i. especially without manual

Intermediate steps, can be performed.

According to a further embodiment of the present invention, in the step of charging and / or in the step of the evaluation, a temperature control of the material to be analyzed, the catalytic material and / or the

Indicator material. Such an embodiment of the present invention

Invention offers the advantage that different sub-steps of the polymerase To be able to use chain reaction different reaction temperatures, so that the individual sub-steps of this polymerase chain reaction can proceed in an optimal environment for each case environment. Furthermore, a method for producing an analysis unit according to a variant presented here is proposed herein, the method comprising the following steps:

Providing the cover element with the at least one

Deckelausnehmung, wherein in the region of the Deckelausnehmung

Cover member having a through hole, the bottom member having at least one bottom recess, the film and a

Probe carrier, as a probe at least one indicator material for

Identification of a biochemical material, wherein the

Indicator material on the probe carrier has a fixed state of aggregation;

Placing the probe carrier in the bottom recess;

Covering the lid recess with the foil; and

Forming the channel region around a fluid in the bottom recess of

Initiate and / or dissipate reaction chamber, the forming by placing the bottom recess opposite to the

Deckelausnehmung done to form the reaction chamber.

Such an embodiment of the present invention offers the advantage of being able to produce an analysis unit for the particularly cost-effective and rapid identification of a biochemical material.

The present invention further provides a control device which is designed to implement or implement the steps of a variant of a method presented here in corresponding devices. Also through this

Embodiment variant of the invention in the form of a control unit, the

Invention underlying task to be solved quickly and efficiently.

In the present case, a control device can be understood as meaning an electrical device which processes sensor signals and outputs control and / or data signals in dependence thereon. The control unit may have an interface, which may be formed in hardware and / or software. At a For example, in terms of hardware, the interfaces may be part of a so-called system ASIC, which performs various functions of the system

Control unit includes. 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 software modules that are available 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

Hard disk space or an optical storage can be stored and used to carry out the method according to one of the embodiments described above, when the program product is executed on a computer or a device. In particular, the approach presented here creates a computer program product with program code for carrying out or controlling the steps of a method presented here when the program product is executed on a device.

The present invention further provides an apparatus configured to perform the steps of a variant of a method presented herein in corresponding apparatus. Also through 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. The invention will now be described by way of example with reference to the accompanying drawings. Show it:

1A is a plan view of an analysis unit according to a

Embodiment of the present invention;

1 B is a cross-sectional view of an analysis unit according to the in FIG.

1A represented an embodiment of the present invention;

FIG. 2 is a plan view of an analysis unit according to another embodiment of the present invention; FIG.

FIG. 3 is a cross-sectional view of an analysis unit according to the embodiment shown in FIG.

2 illustrated embodiment of the present invention;

4 is a plan view of an analysis unit according to another embodiment of the present invention;

5A and 5B are top views of analysis units according to further embodiments of the present invention;

6A is a cross-sectional view of a probe carrier according to a

Embodiment of the present invention;

6B is a cross-sectional view of a probe carrier according to a

another embodiment of the present invention;

Fig. 7 is a schematic representation of an analysis device with a schematic representation of a in the analysis device

incorporated analysis unit;

8 is a flowchart of an embodiment of a method for operating an analysis unit; and

9 is a flowchart of an embodiment of a method for manufacturing an analysis unit. 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 is made to a repeated description of this

Elements is omitted.

In the biochemical approach presented here, the DNA microarray (as an example of a probe carrier) is directly in a PCR reaction chamber

involved. The DNA probes immobilized on the DNA microarray actively participate in the PCR and thus immobilize specifically

Amplification product (i.e., the biochemical material). The

Surface-bound products (or intermediates) from a biochemical process (such as PCR) can be detected and / or evaluated either in real time or after the reaction.

1A shows a top view of an analysis unit 100 according to an exemplary embodiment of the present invention. FIG. 1B shows a

Sectional view of the embodiment of the present invention shown in FIG. 1A along A of FIG. 1A. In this case, the analysis unit

100 only a single PCR reaction chamber 1 10 on.

The analysis unit 100 comprises a plurality of layers, as can be seen from FIG. 1B. A layer structure consists of a first polymer substrate as cover element 130, which has a cover recess 135 and a

Through hole 137, wherein on the underside of the cover member 130, a deflectable polymer membrane is attached as a film 140. This film 140 may, for example, be connected in a fluid-tight manner to the cover element 130 so that contact between a medium that can be moved through the through-hole and not reach a region outside the film 140.

Planar parallel to this structure of the cover member 130 is a bottom member 150, for example in the form of a second polymer substrate having in a Bodenelementausnehmung 152, the PCR reaction chamber 1 10 with a DNA microarray as a probe support 155. On the microarray 155 are different oligonucleotides as probes 157 in the form of individual Spots immobilized. For filling and emptying, a microfluidic channel 160 (which is separated, for example, by the reaction chamber 110 into two sub-channels 160a and 160b) is connected to the chamber 110. Both channels 160a and 160b each have a controllable valve 165 to communicate with a PCR reaction mix (ie with material to be analyzed and / or with

Catalyst material which is used as auxiliary material for carrying out the

biochemical reaction is required) to keep filled chamber 1 10 closed during the (biochemical) reaction. In a defined region 175, in which the reaction chamber 110 is arranged, thermal energy for carrying out a PCR can be introduced and removed by an analysis device described in more detail below.

During the PCR, certain DNA motifs of a template DNA are first amplified in the liquid phase. Formed PCR products can now bind to section-specific, immobilized oligonucleotides. By

Identification of the spots to which a PCR product has been attached make statements about the presence / absence of certain DNA motifs possible.

FIG. 2 shows a top view of an analysis unit 100 according to an embodiment of the present invention. Fig. 3 shows a

Sectional view along the section line A of shown in Fig. 2

Embodiment of the analysis unit 100, the two on the sub-channel 160b fluidly coupled to each other PCR reaction chambers 1 10 and 210 has. The second reaction chamber 210 can be formed analogously to the (first) reaction chamber (except for the presence of the probe carrier 155).

For example, to form the second reaction chamber 210, the cover element 130 may have a further cover recess 235 which is fluidically connected to an external environment of the analyzer 100 via a further through-hole 237. The film can also close the second lid recess 235 in a fluid-tight manner on a side opposite the further through-hole. The bottom element 150 may have a further bottom recess 252 through which the second reaction chamber 210 is formed.

In this exemplary embodiment, two defined temperature ranges 175 and 220 exist in which a constant, but different temperature is set over the entire time of the PCR. This will be the Requirements for a temperature control unit of an analysis device compared to an analysis device for operation of the analysis unit 100 according to the embodiment illustrated in Figures 1 A and 1 B, in which the temperature between -60 ° C and -95 ° C in a time window of minutes are varied must (eg by means of Peltier element). In order to expose the PCR reaction mix to different temperatures, in the embodiment illustrated in Figures 2 and 3, the reaction volume (ie, the fluid having the material to be analyzed and / or the catalytic material) is pushed back and forth between the two chambers by means of the deflectable polymer membrane 140 , This reciprocating movement can take place, for example, by a pressure which exerts on the film 140 via the through-holes 137 or 237 on the respective reaction chambers 1 10 and 210, wherein the bulging of the film into these reaction chambers 1 10 or 210 let out the fluids located in the respective reaction chambers.

An important goal of the approach presented here can be seen as a possibility for suitable structures and processes for the

Performing a PCR in a polymeric multilayer composite. This achieves a shortening of the process time and a reduction of process steps, for example, in the detection of nucleic acids.

In this case, a polymeric layer structure of the analysis unit 100 with an integrated DNA microarray 155 can be used. The DNA microarray 155 is present either directly in a PCR reaction chamber 110 or in a separate array chamber. By pushing back and forth the reaction volume (i.e., the fluid with the material to be analyzed or the catalytic material) between chambers 110 and 210, which are at different temperatures, a PCR reaction is carried out. The immobilized

Oligonucleotides as probes 157 of the microarray 155 actively participate in the PCR reaction so that DNA sequences are located at certain locations of the array 155.

The approach presented here results in the following advantages as a biochemical reaction using the example of a PCR:

- The PCR product does not need to be rebuffered before hybridization. - The LoC system no longer needs a buffer for hybridization.

The described structures and methods enable a faster process flow together with a simplified process control.

- Shortening the total duration of proof of genetic characteristics.

In the presented process, PCR products are already being used during the

PCR spatially resolved and detectably immobilized at specific spots of the microarray.

- Use of a surface-sensitive measuring method.

According to a further exemplary embodiment of an analysis unit 100, the reaction takes place in a system of three PCR chambers 1 10, 210 and 310, which are fluidically interconnected via sub-channels 160, as described in US Pat

Top view of Fig. 4 is shown. Now the three

Analysis chambers 1 10, 210 and 310 constructed analogously to each other, wherein in the first two analysis chambers 1 10 and 210 each have a probe carrier 255th

is arranged. As a result, three different temperature-controlled regions / chambers can be realized for a 3-step PCR, for example for three-step PCR reactions (eg annealing temperature 175 = 50 ° C.-65 ° C.,

Extension temperature 220 = 72 ° C; Melting temperature 320 = 95 ° C). The array 155 or 255 can in this case either in the chamber 1 10 on

Annealing temperature of 175 in the chamber 210 on

Extension temperature 220 is to be positioned. Furthermore, the first reaction chamber 1 10 and the second reaction chamber 310 to a

Temperature between 55 and 72 ° C tempered (Anlagerungs- and

Extension temperature), and the intermediate chamber 210 may be tempered to the melting temperature.

For a 2-step PCR, the reaction volume may first be reciprocated (using sub-channel 160b) between, for example, chambers 110 and 210 for between 1 to 40 cycles, and subsequently (under

Use of the sub-channel 160c) between the chambers 210 and 310 between 1 to 40 cycles, in which case, for example, contrary to the illustration of FIG. 4, only the chamber 310 contains the (second) array 255. FIG. 5A shows a top view of an analysis unit 110 according to a further exemplary embodiment of the present invention, in which a Reaction chamber 1 10 (array chamber) contains the array 155. The chamber 1 10 is located either in the temperature range 175 of the annealing temperature or extension temperature 220 (as, for example, in the

Top view of FIG. 5B can be seen). This embodiment allows PCR to be initially performed in the PCR chambers 410, 210, or 310 without the array 155. After amplification, the reaction mixture is then transferred into the array chamber 110, so that the PCR products can be specifically bound to oligonucleotides 157. The attachment can be by means of hybridization or primer extension reaction, for example by moving the reaction volume back and forth between a chamber

Melting temperature and the array chamber 1 10.

According to another embodiment, the chamber inlet 160a and outlets 160b of a fluidic layout may be provided with check valves 165 as shown in FIGS. 2, 4, 5A and 5B. This will be added

Volume displacement a (single) pumping direction given what the

Actuation of the fluid simplified.

FIGS. 6A and 6B each show a structure to protect the surface of the array 155 (specifically, the spots of the immobilized oligonucleotides as probes 157). As for the transfer of the PCR reaction volume from a PCR chamber 1 10 in another (as shown in FIG. 2 and FIG. 4), the polymer membrane 140 is pneumatically deflected, while in the direction of the bottom of the

Bodenausnehmung 152 is pressed, possibly in the

Array format 155 immobilized oligonucleotides 157 are damaged. When

As a countermeasure, the spots of the array 155 may be immobilized in either wells 610 or on a microscopically rough surface 620.

Oligonucleotides 157 can be used with all immobilization chemistries known in the art, such as 3-aminopropyltriethoxysilane (APTES), 1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide (EDC), 1, 4-phenylene diisothiocyanate (PDITC). , s-SIAB, s-MBS, s-GMBS or s-MBP on a variety of materials on the probe carrier 155 are fixed. The arrays 155 may be either directly in one

Polymer substrate (ie, the bottom member 130) are immobilized or on a Substrate 155 are spotted, which is then introduced and fixed in a second step in a PCR chamber.

FIG. 7 shows a cross-sectional illustration of an exemplary embodiment of an analysis device 700 for operating an analysis unit 100 according to an exemplary embodiment of the present invention with a polymer

Layer structure. The analysis device 700 has on its upper side a Heizbzw. Tempering (tempering) 710 and on the underside of a detection unit 720 (evaluation unit) is mounted. The arrangement of tempering element 710 and detection unit 720 can also be reversed. The tempering element 710 may also comprise a plurality of subunits, in which the adjacently arranged regions of an analysis unit 100 are heated to different temperatures at the same time. Thermal energy for performing a temperature controlled PCR can be obtained from the

Tempering 710 be introduced inter alia by the use of tempered heating fingers, Peltier elements, infrared radiators or convection. In order to hold the analysis unit 100, a holder 730 can also be provided which fixes the analysis unit 100, for example, on a surface of the temperature control unit 710 during the operation of the analyzer.

The PCR products attached to a microarray 155 can be used either during or after the reaction with various methods in the

Detection unit 720 can be identified:

1 . Fluorometric detection. During PCR, fluorophores are incorporated into the resulting PCR products. After excitation of these fluorophores (eg with evanescent fields, confocal or with transmitted light), the emitted fluorescence signals can be measured by means of CCD cameras, CMOS chips or photomultipliers.

2. Detection of surface plasmons. For example, oligonucleotides 157 are immobilized on gold spots. After or during PCR, surface plasmons in the spots are excited. Depending on the amount of near-surface DNA molecules, the plasmon resonance frequency shifts. By measurement and evaluation of the Spots emitted intensities and / or frequencies can be up

Binding events are deduced.

The reaction mixture contains the components polymerase, reaction buffer, PCR primer and nucleotides and reaction-improving components such. BSA and / or Tween. If biotin-dUTP nucleotides are used in the PCR, the resulting PCR products are labeled with biotin molecules to which fluorophore streptavidin conjugates can be added in a further step. Alternatively, fluorophore-labeled primers can be used in the PCR so that the generated PCR products are labeled directly with a fluorophore. For the

Performing an asymmetric PCR, the two PCR primers can be used in different concentrations. The necessary structures in the polymer substrates can be produced for example by milling, injection molding, hot stamping or laser structuring. The microarray 155 can either be formed directly in the polymer or be introduced into the polymer layer structure as an insert, for example made of glass.

Material examples:

Polymer substrate (for example for lid 130 and bottom element 150):

Thermoplastics (eg PC, PP, PE, PMMA, COP, COC, PEEK)

Polymer membrane (film 140): Elastomer, thermoplastic elastomer TPU, TPS, thermoplastics, hot-melt adhesive films, sealing films for microtiter plates, latex

Exemplary formation of the biomolecules:

Length of oligonucleotides 157: 5-100 nucleotides

- Linker molecules: thiol groups, amino groups, gold, glutharaldehyde,

Acrydites, for example via a carbon chain connected to the oligonucleotides

- PCR primer: length between 5 and 100 nucleotides, whereby the

Melting temperature of the two primers can differ greatly

Polymerase: hot-start polymerases, proof-reading polymerases

- Diameter of a spot: 1-500 μηη

Exemplary dimensions of the embodiments:

Thick polymer substrate: 0.5 to 5 mm

- Channel diameter in polymer substrates: 10 μηη to 3 mm Thick polymer membrane: 5 to 500 μm

Volume of the cavities in the polymer substrates: 1 mm 3 to 1000 mm ³

Exemplary pressures for the actuation of a polymer membrane:

- 0.2 bar - 2 bar

The invention can be used for analytical systems, in particular for microfluidic lab-on-chip systems for environmental analysis or medical diagnostics.

8 shows a flow chart of an embodiment of a method 800 for operating an analysis unit. The method 800 includes a step 810 of providing the analysis unit and a step 820 of the

Loading the analysis unit with a material to be analyzed and / or with a catalytic material for carrying out a reaction in the analysis unit. Finally, the method 800 includes a step 830 of evaluating a change in the indicator material on the probe carrier.

9 shows a flow chart of an exemplary embodiment of a method 900 for producing an analysis unit. The method 900 comprises a step 910 of providing the lid element with the lid having at least one lid recess, wherein in the region of the lid recess

Cover element has a through hole, the bottom member having at least one bottom recess, the film and a probe carrier having at least one indicator material for identifying a biochemical material as a probe, wherein the indicator material on the

Probe carrier has a solid state of aggregation. Further, the method 900 includes a step 920 of placing the probe carrier in the bottom recess and a step 930 of covering the probe

Lid recess with the foil. Finally, the method 900 includes a step 940 of forming the channel region around a fluid in the

Bottom recess of the reaction chamber to initiate and / or dissipate, wherein the forming is done by placing the bottom recess opposite the Deckelausnehmung to form the reaction chamber. 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, method steps according to the invention can be repeated as well as carried out in a sequence other than that described.

If an exemplary embodiment comprises a "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

1 . Analysis unit (100) for carrying out a polymerase chain reaction, wherein the analysis unit (100) has the following features:

a lid member (130) having at least one lid recess (135); - A bottom member (150) having at least one bottom recess (152), wherein the bottom recess (152) of the lid recess (135) is arranged opposite to form a reaction chamber (1 10);

a film (140) which is arranged at least in the region of the cover recess (135) between the cover element (130) and the bottom element (150);

at least one channel (160a, 160b) formed between the lid member (130) and the bottom member (150) for introducing and / or discharging a fluid into the bottom recess (152) of the reaction chamber (110); and

a probe carrier (155) arranged in the bottom recess (152) and serving as probe (157) at least one indicator material for

Identification of a biochemical material, wherein the indicator material on the probe carrier (155) has a solid

Has state of aggregation.

2. Analysis unit (100) according to claim 1, characterized in that the film (140) on the lid member (130) is fixed, in particular wherein the film (140) one of the passage opening (137) opposite opening of the lid recess (135) closes fluid-tight and / or wherein in the region of the cover recess (135) the cover element (130) has a passage opening (137).

Analysis unit (100) according to one of the preceding claims, characterized in that the cover element (130) at least one further cover recess (235) and the bottom element (150) at least a further bottom recess (252), wherein the further

Cover recess (235) of the further bottom recess (252) is arranged opposite and wherein the bottom recess (152) and the further bottom recess (252) by means of the channel (160a, 160b) are fluidly coupled.

Analysis unit (100) according to claim 3, characterized in that in the further bottom recess (252) another probe carrier (255) is arranged, which has as further probe (257) at least one further indicator material for the identification of another biochemical material, wherein the further indicator material on the other

Probe carrier (255) has a fixed state of aggregation.

Analysis unit (100) according to one of the preceding claims, characterized in that the bottom recess (152) separates the channel (160a, 160b) into at least two subchannels, wherein at least one subchannel (160a, 160b) has a fluidic connection to an external environment of the analysis unit (15). 100), in particular wherein at least one partial channel (160a, 160b) has a valve (165).

Analysis unit (100) according to claim 5, characterized in that the sub-channels at one of the bottom recess (152) facing away from each other are fluidly coupled or coupled, in particular wherein the sub-channels components of an annular channel (160a, 160b).

Analysis unit (100) according to one of the preceding claims, characterized in that the probe carrier (155) is formed and arranged such that during the polymerase chain reaction, a reaction of a substance with the indicator material for the identification of the biochemical material is executable.

An analysis device (700) for operating an analysis unit (100) according to one of the preceding claims, wherein the analysis device (700) has at least the following features:

a holder (730) for placement and / or mounting of

Analysis unit (100) during operation of the analysis device; a temperature control unit (710) for controlling the temperature of a fluid or a solid in the analysis unit (100) held by the holder; and

an evaluation unit (720) for evaluating a change in the indicator material.

9. Analysis device (700) according to claim 8, characterized in that the tempering unit (710) is designed to simultaneously bring different sections of the analysis unit (100) to a different temperature.

10. The method (800) for operating an analysis unit (100) according to one of claims 1 to 7, wherein the method (800) comprises the following steps:

- providing (810) the analysis unit (100);

Loading (820) the analysis unit with a material to be analyzed and / or with a catalytic material for carrying out a reaction in the analysis unit (100); and

Evaluating (830) a change in the indicator material on the probe carrier (155).

1 1. A method according to claim 10, characterized in that the steps of loading and evaluating are carried out at least partially simultaneously.

12. The method according to claim 10 or 1 1, characterized in that takes place in the step of applying (820) and / or in the step of evaluating (830) a temperature control of the material to be analyzed, the catalytic material and / or the indicator material.

13. Method (900) for producing an analysis unit (100) according to one of claims 1 to 7, wherein the method (900) comprises the following steps:

Providing (910) the cover element with the at least one cover recess (135), the cover element (130) having a passage opening (137) in the region of the cover recess (135), of the bottom element (150) having at least one bottom recess (152), the foil (140) and a probe carrier (155) having as probe (157) at least one indicator material for identifying a biochemical material, wherein the indicator material on the probe carrier (15) 155) has a fixed state of aggregation;

Placing (920) the probe carrier (155) in the bottom recess (152);

- Covering (930) of the lid recess (135) with the film (140); and

Forming (940) the channel region (160a, 160b) around a fluid in the

Bottom recess (152) of the reaction chamber (1 10) to initiate and / or dissipate, wherein the forming by arranging the

Bottom recess (152) opposite the lid recess (135) takes place to form the reaction chamber (1 10).

14. Control unit with units which are designed to control and / or implement the steps of a method (800, 900) according to one of claims 10 to 13.

Computer program product with program code for carrying out or triggering the steps of the method (800, 900) according to one of claims 10 or 13, when the program product is executed on a device.