WO2015162060A1 - Dispositif microfluidique et procédé d'analyse d'un échantillon de matière biologique - Google Patents

Dispositif microfluidique et procédé d'analyse d'un échantillon de matière biologique Download PDF

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Publication number
WO2015162060A1
WO2015162060A1 PCT/EP2015/058354 EP2015058354W WO2015162060A1 WO 2015162060 A1 WO2015162060 A1 WO 2015162060A1 EP 2015058354 W EP2015058354 W EP 2015058354W WO 2015162060 A1 WO2015162060 A1 WO 2015162060A1
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Prior art keywords
sample
chamber
target molecules
reaction
amplification section
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PCT/EP2015/058354
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German (de)
English (en)
Inventor
Jochen Rupp
Jochen Hoffmann
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Robert Bosch Gmbh
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Publication of WO2015162060A1 publication Critical patent/WO2015162060A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/088—Channel loops
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components

Definitions

  • the present invention relates to a microfluidic device for analyzing a sample of biological material, to a method for
  • PCR polymerase chain reaction
  • a microfluidic device for analyzing a sample of biological material a method for analyzing a sample of biological material, a
  • a system 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.
  • a spatial, thermal and fluidic separation of the two individual functions can be achieved in order to enable advantageous processing.
  • 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.
  • a reaction volume of a sample between chambers which may be at different temperatures, for example, a PCR can be carried out.
  • the reaction volume can be brought into contact with the DNA microarray.
  • Probe microarrays interact with strands of a PCR product and fix the same.
  • an improved array PCR can be made possible.
  • Method according to embodiments of the invention in a faster process flow and with a simplified process management are made possible.
  • 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.
  • Read reaction or hybridization, with or without washing of the microarray can in this case in particular at different times parallel to or
  • 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.
  • 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
  • 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
  • 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.
  • a liquid to be analyzed typically a liquid or liquefied patient sample, e.g.
  • 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
  • Valves may be arranged in the fluid lines.
  • cells of a sample can be lysed before the PCR and subsequently the DNA can be purified.
  • 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.
  • Reaction chamber and the at least one detection chamber on respective
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment offers the advantage that the microfluidic device can also meet different or changing requirements of sample analyzes.
  • 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.
  • a second reaction chamber may be formed to allow the primer hybridization of denatured target molecules of the sample.
  • a third reaction chamber may be formed to allow the elongation of primary hybridized target molecules of the sample.
  • the at least one device for transport for repeatedly conveying the sample between the reaction chambers may be formed.
  • the sample or a sample volume can be located both between the reaction chambers and between the amplification section and the at least one
  • reaction chambers may be fluidly connected in series in the amplification section.
  • Embodiment offers the advantage that the sample processing can be further accelerated because, inter alia, each reaction chamber is individually preheated.
  • the method may be advantageously carried out in conjunction with an embodiment of the aforementioned microfluidic device to analyze a sample of biological material.
  • the steps may be repeated for each cycle of the polymerase chain reaction.
  • 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
  • a device in the form of a device, the object underlying the invention can be solved quickly and efficiently.
  • a device in the form of a device, the object underlying the invention can be solved quickly and efficiently.
  • 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.
  • the interfaces can be part of a so-called system ASIC, for example, which contains a wide variety of functions of the device.
  • the interfaces are their own integrated circuits or at least partially consist of discrete components.
  • 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
  • Figures 1 to 4 are schematic representations of a microfluidic device with fluid conveying directions according to embodiments of the present invention
  • FIG. 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
  • Fig. 7 is a diagram showing intensity signals for various amplified and hybridized DNA segments.
  • FIG. 1 shows a schematic representation of a microfluidic device
  • 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
  • the microfluidic device 100 is For example, a so-called chip lab or lab-on-chip (LOC).
  • LOC lab-on-chip
  • 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.
  • the denaturation chamber 111 is designed to perform or permit denaturation of target molecules of the sample.
  • Hybridization chamber 113 is designed to perform primer hybridization of denatured target molecules of the sample.
  • Elongationshunt 112 is formed to a elongation of
  • primerhybridformaten target molecules of the sample perform or to
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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 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.
  • 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
  • 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
  • the first fluid conveying direction A in this case runs clockwise from the
  • Fluid conveying direction B runs counterclockwise from the
  • Fluid conveying directions A and B will be discussed in more detail below.
  • FIG. 1 shows a fluidic structure of the FIG.
  • the structure comprises the three reaction chambers 111, 112 and 113, in which, with alternate fluid or liquid transport of a liquid
  • 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.
  • 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
  • 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.
  • 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).
  • PCR polymerase chain reaction
  • a combined amplification and detection reaction is defined in particular by fluidic, thermal and biochemical protocols.
  • a sample solution in a PCR mix in the denaturation chamber 111 becomes
  • 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.
  • 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.
  • 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.
  • the reaction volume is cyclically brought into contact with the microarray in the detection chamber 130.
  • 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.
  • Fluid conveying direction B via the same path in the opposite direction via the reaction chambers 113 and 112 back into the Denaturierungshunt 111 or promoted.
  • 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.
  • 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. 2
  • 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
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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
  • Detection chamber 130 thus takes place, for example, between once and 40 times.
  • Embodiments of the present invention received.
  • Microarray chamber a washing solution are rinsed while the sample volume is in one of the reaction chambers 111, 112 and 113.
  • substances which inhibit readout reaction such as fluorophores, can be washed away.
  • the rinse solution may be a PCR buffer, which may comprise, for example, all the components mentioned in the previous section.
  • 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
  • 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 TM 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
  • 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
  • the method 500 may be described in conjunction with
  • microfluidic device such as one of
  • 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
  • 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.
  • 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.
  • steps 510 to 550 are repeated or re-executed in each cycle of the polymerase chain reaction.
  • the method 500 further comprises a step 560 of completing a molecular staining process of
  • Step 560 of completing the molecular staining process will be described below in particular
  • 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.
  • Fig. 6 are a first partial view A, a second
  • first partial view A a product strand 601 and biotin
  • Desoxvuridine triphosphates 602 and biotin-dUTPs respectively.
  • a PCR reaction mix also contains biotin-dUTPs 602.
  • these particular nucleotides i.e. H. the biotin-dUTPs 602, during the reaction or PCR in the
  • Product strand 601 installed.
  • 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.
  • the strand 604 connected to the probe of the microarray 603 is fluorescently stained.
  • 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.
  • an amount of dUTPs used in the PCR is an amount of dUTPs used in the PCR, a length of PCR
  • a PCR reaction mix can only contain biotin-dUTPs, but not deoxythymidine triphosphates or dTTPs.
  • biotin-dUTPs and dTTPs are used at a ratio of five to one to one to five.
  • 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
  • Amplification be made unusable in another PCR. This is an advantageous biochemical measure to combat a series of DNA contaminations.
  • a reagent which contains sodium phosphate buffer in one
  • Tween 80 or Tween 20 may be added.
  • 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
  • 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
  • washing solution or washed successively with two washings.
  • the following parameters can be used: 100 millimoles, sodium phosphate buffer, pH 7.2, between 0.1 and 10 minutes
  • 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
  • Fluorophores accumulate on a two-dimensional surface to provide well-detectable signals against the background.
  • 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,
  • microarrays 603 can be improved because a number of fluorophores per bound DNA segment can be increased, for example, more than doubled.
  • 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
  • first signals 710 are higher than a detection limit defined at 1000 counters than second signals 720.
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un dispositif microfluidique (100) pour l'analyse d'un échantillon de matière biologique. Le dispositif microfluidique (100) comporte une section destinée à l'amplification (110) comprenant au moins une chambre de réaction (111, 112, 113) pour la réalisation d'une réaction en chaîne par polymérase présentant une multitude de cycles pour une amplification de l'échantillon. A cet effet, la ou les chambres de réaction (111, 112, 113) sont conçues pour permettre une dénaturation de molécules cibles de l'échantillon, une hybridation des molécules cibles dénaturées de l'échantillon avec des amorces, et un allongement des molécules cibles de l'échantillon hybridées avec les amorces. Le dispositif microfluidique (100) comporte au moins une chambre de détection (130) pour la mise en oeuvre répétée de réactions de détection sur les molécules cibles dénaturées de l'échantillon provenant de différents cycles de la réaction en chaîne par polymérase. La ou les chambres de détection (130) sont reliées fluidiquement à la section destinée à l'amplification (110). Le dispositif microfluidique (100) comporte au moins un dispositif de transfert (120) pour le transfert répété de l'échantillon entre la section destinée à l'amplification (110) et la ou les chambres de détection (130).
PCT/EP2015/058354 2014-04-25 2015-04-17 Dispositif microfluidique et procédé d'analyse d'un échantillon de matière biologique WO2015162060A1 (fr)

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DE102014221616.8A DE102014221616A1 (de) 2014-04-25 2014-10-24 Mikrofluidische Vorrichtung sowie Verfahren zum Analysieren einer Probe biologischen Materials
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JPWO2017213080A1 (ja) * 2016-06-06 2019-04-11 株式会社ニコン 流体デバイス、システム、試料物質の検出方法および試料物質の精製方法

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