WO2019081259A1

WO2019081259A1 - Reaction carrier for a microfluidic device and method for determining a nucleotide sequence

Info

Publication number: WO2019081259A1
Application number: PCT/EP2018/078140
Authority: WO
Inventors: Jochen Hoffmann
Original assignee: Robert Bosch Gmbh
Priority date: 2017-10-23
Filing date: 2018-10-16
Publication date: 2019-05-02
Also published as: DE102017218849A1

Abstract

The invention relates to a reaction carrier (100) for a microfluidic device (200) for determining a nucleotide sequence, comprising at least one first recess (101) and a second recess (102), in which first scavenger primers (121) are immobilised on a solid phase in the first recess (101), and second scavenger primers (122) are immobilised on a solid phase (110) in the second recess (102), the first scavenger primers (121) being different from the second scavenger primers (122). The invention also relates to such a microfluidic device (200) and to an associated method (500) for determining a nucleotide sequence.

Description

Description title

Reaction carrier for a microfluidic device and method for

Determination of a nucleotide sequence

State of the art

Known microfluidic devices, also called lab-on-a-chips, can be used to detect DNA-containing substances, for example pathogens, via clear gene signatures, for example via so-called sequencing-by-synthesis methods (inter alia Bentley et al Nature 456 (7218): 53-59 and Ju et al. (26 October 2006) "Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators"; PNAS 2006 103 (52) 19635-19640;

doi: 10.1073 / pnas.0609513103). The detection reactions are common that nucleotide sequences of these substances are queried in the context of the so-called genotyping by means of complementary fluoreszenzgelabelter DNA probes. In contrast, genotyping can only detect the gene sequences for which the device also has a corresponding probe. However, pathogens change genetically over time, often in the course of resistance training. Therefore, the probes held in the device must be adapted again and again to cover these changes as well as newly discovered pathogens. Disclosure of the invention

Advantages of the Invention Against this background, the invention relates to a reaction carrier for a microfluidic device for determining a nucleotide sequence. The reaction carrier comprises at least a first recess and a second recess, wherein in the first recess first capture primer and in the second recess second capture primer are immobilized on a solid phase. The first capture primers are different from the second capture primers.

The reaction carrier may in particular be a substrate, for example a

Plastic substrate include. Under a recess can be understood in particular a depression in a surface of the reaction carrier. The nucleotide sequence is in particular a sequence of

Nucleotides of a nucleotide strand or nucleic acid fragment of a nucleotide strand

Deoxyribonucleic acid or ribonucleic acid, for example one

Nucleotide strand of a pathogen or a human or animal cell. Under a pathogen is to understand a pathogen,

in particular a virus, a bacterium or a unicellular. Under one

Determination of a nucleotide sequence may in particular

Sequencing methods are understood as described above, in particular a sequencing-by-synthesis method. Both

Capture primers are, in particular, nucleotide strands which are designed to bind the nucleotide strands to be sequenced.

Preferably, the capture primers are further designed for amplification of the nucleotide strands to be sequenced, in particular for one

Reproduction via a polymerase chain reaction (PCR). The reaction support advantageously allows the amplification of the nucleotide strands to be sequenced, in particular by carrying out a PCR, on the reaction support, ie directly on a solid phase, to what

immediately afterwards a sequencing on the solid phase can be carried out. In such a solid-phase PCR, the resulting PCR product is attached to the immobilized capture primer. The too sequencing nucleotide strands are immobilized during PCR by binding to the capture primers and thus advantageously provided at a given location, namely at the site of the solid phase, for subsequent sequencing. The immediate immobilization of the

Nucleotide strands also allow easy removal of no longer required reaction components, especially by rinsing the solid phase. Furthermore, it is of particular advantage that no specific probes for detecting the nucleotide strands must be provided, but identification via the sequencing of the nucleotide strands bound to the capture primers is possible. In particular, it is advantageously possible to recognize known DNA signatures and also point mutations in such signatures. The immobilized and different capture primers also advantageously allow parallel and spatially resolved sequencing and identification of different nucleotide strands.

The reaction carrier may preferably comprise further recesses with further immobilized capture primers, wherein the further capture primers may be partially identical and in part different from each other. This advantageously increases an effectiveness and efficiency or a parallelism of the determination of the nucleotide sequences.

A solid phase can be understood as meaning a material, a substance or a composition of materials and / or substances in a solid state of matter, including gels, in particular agarose gels or hydrogels. The solid phase may be connected to the reaction carrier, in particular, the

Solid phase to be part of the reaction carrier. The solid phase preferably comprises at least part of a wall or a bottom of the first and / or the second recess. As a result, the catcher primers are advantageously immobilized in the recesses.

Alternatively or additionally, the solid phase can comprise particles, in particular magnetic particles, which can advantageously be immobilized with respect to the solid phase with the aid of a magnet. In this case, magnetic particles can be understood as meaning particles having a magnetic or magnetizable material, for example paramagnetic polymer particles. The use of particles as a solid phase has the advantage that at

Use of a liquid sample with binding to be contained therein

Nucleotide strands a contact surface between the solid phase and the sample is greater than when using a planar phase, which provides for increased and thus improved reaction kinetics due to shortened diffusion paths. The use of magnetic particles as a solid phase also has the advantage that, on the one hand, the particles can be retained by a magnetic force at well-defined and thus easily distinguishable locations in the microfluidic device. On the other hand, the immobilized nucleotide strands can be transported to any location via magnetic actuation, for example for subsequent processing or analysis. Furthermore, it is advantageous that rinsing of the particulate solid phase can be carried out more effectively in comparison to a planar solid phase

According to a particularly advantageous embodiment of the invention, the solid phase comprises porous material, wherein the first and / or the second capture primer are immobilized in pores of the porous material. This has the advantage that the surface for the immobilization of capture primers and thus sequenceable nucleotide strands is significantly increased due to the pores.

In an advantageous development of the invention, at least one of the recesses contains further primers for the determination of the nucleotide sequence in unbound form, in particular in dried form. The further primers are preferably designed to support the PCR for the amplification of the nucleotide strands which bind to the capture primer. Preferably, the further primers are at least partially identical to primers for the PCR of these nucleotide strands. In one embodiment, the further primers in the first recess or in the second recess are at least partially identical to the first or the second catcher primer. For example, a sequence of the further primer in the first recess or in the second recess with a

Partial sequence starting at the 3 'end of the first or second capture primer identical. In a particularly advantageous embodiment of the invention are in at least one of the recesses further primer for the implementation of a

Amplification reaction before, especially in dried form. Under an amplification reaction is a method of amplification of

Nucleotide strands to understand, for example, an isothermal

Amplification reaction or a PCR. Preferably, the further primers are designed for a multiplication of the nucleotide strands to be sequenced, that is to say, in particular, for the nucleotide strands to be sequenced and for the first or second capture primers. In the case of a PCR as an amplification reaction, the further primers may be reverse primers or forward primers if the primers are forward primers or reverse primers. Furthermore, further reagents for carrying out the amplification reaction, in particular a PCR, can be present in at least one of the recesses, in particular in dried form, for example additives for the amplification reaction.

The subject of the invention is also a microfluidic device with a reaction carrier according to the invention. For example, the reaction carrier is detachably arranged in the microfluidic device, for example via a latching mechanism or Einklippmechanismus. Alternatively, the

Reaction carrier also be connected via a bond with the microfluidic device, which advantageously allows a particularly fluid-tight connection. The reaction carrier can thus advantageously according to the

Production and immobilization of the first and second capture primer can be introduced into the device.

According to a particularly advantageous embodiment of the invention, the microfluidic device comprises an area for pre-amplification of the nucleotide strands to be sequenced. This area may be

in particular act around a chamber of the device, wherein preferably a fluidic connection between the chamber and the reaction carrier can be interrupted, for example by a valve. Under one

Pre-duplication is a duplication of the sequences to be sequenced

Nucleotide strands to understand before the nucleotide strands to be sequenced are brought into contact with the reaction support, for example a Duplication via PCR, especially as part of a nested PCR. This has the advantage that a large number of the to be sequenced

Nucleotide strands can be generated in the microfluidic device. Optionally, in this area, a dilution, in particular with a buffer solution, take place, in particular after the pre-duplication.

Preferably, the microfluidic device comprises a to the

Reaction support adjacent region, wherein the region for receiving a magnet is formed such that a magnetic field of the magnet for fixing a magnetic or magnetizable solid phase described above in the recesses may extend into one or more of the recesses. The magnet can be a permanent magnet or an electromagnet, which can generate a magnetic field suitable for the manipulation of the particles. For example, the reaction carrier may have a recess for receiving a magnet. The device can in addition to the preferred latching or

Einklippmechanismus a region, in particular a recess, for receiving the magnet.

The invention further provides a method for determining a nucleotide sequence with a reaction carrier according to the invention. In a first step of the process, first capture primers of the first

Recess and second capture primer in the second recess of the

Reaction carrier immobilized, wherein the first capture primer are different from the second capture primer. In a second step become

sequencing nucleotide strands bound to the first and / or second capture primers. Before that, the nucleotide strands to be sequenced may have been amplified in an optional step, in particular by performing a PCR. In a third step, the sequence of bound

Nucleotide strands determined, in particular via a sequencing-by-synthesis method.

For the advantages of the method according to the invention and the following advantageous developments and refinements, reference is also made to the above-mentioned advantages. Preferably, the immobilizing step comprises binding the

Capture primer to the solid phase of the reaction carrier.

The solid phase can be a mobile solid phase,

for example, preferably magnetic particles, as stated above. In a particularly advantageous embodiment of the method, the binding of the capture primer to the solid phase takes place prior to the immobilization of the solid phase on the reaction support. This has the advantage that the binding of the capture primer to the solid phase can also take place separately from the reaction support and in particular outside the microfluidic device.

According to a particularly advantageous embodiment of the method takes place during the binding of the nucleotide strands to the first and / or second capture primer, a closure of the recesses, in particular by a cover with a liquid. This has the advantage that a separate and transverse contamination-free reaction can take place in each recess. Furthermore, the recesses are advantageously protected against contamination and it is difficult to escape from the recesses. The liquid may in particular be a non-polar liquid, for example an oil.

Brief description of the drawings

Embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description. The same reference numerals are used for the elements shown in the various figures and similarly acting, wherein a repeated description of the elements is dispensed with.

Show it

Figures 1 and 2 an embodiment of the device according to the invention and FIG. 3 shows a flowchart for an exemplary embodiment of the method according to the invention.

Embodiments of the invention

FIG. 1 schematically shows an exemplary embodiment of the microfluidic device 200 according to the invention with an exemplary embodiment of the invention

Reaction carrier 100 according to the invention for determining a

Nucleotide sequence. The reaction carrier 100 comprises in this example a first recess 101, a second recess 102 and a third

Recess 103, which are each designed as depressions in the reaction carrier 100. For example, the depressions 101, 102, 103 each have a volume between 1 picoliter and 100 microliter, preferably between 1 nanoliter and 10 microliter.

All the recesses 101, 102, 103 comprise a solid phase 110, in this example magnetic particles 110. The particles 110 may be

in particular, magnetic microparticles or nanoparticles having an exemplary diameter between 10 nanometers and 500 micrometers, preferably between 100 nanometers and 100 micrometers, such as Dynabeads® or BioMag® and BioMag® "Unconjugated Particles." To the particles 110 in the first recess 101 are first capture primers 121, second capture primers 122 are bound to the particles 110 in the second recess 102 and third capture primers 123 are bound to the particles 110 in the third recess 103. Different capture primers 121, 122, 123 thus enable parallel and spatially resolved detection of different nucleotide strands. Alternatively or additionally, a part of the reaction support 100, in particular a part of a wall 131 or a bottom 132 of one or more

Recesses 101, 102, 103, porous material, wherein in the pore capture primer are immobilized. For example, this part of the

Reaction carrier 100 comprise porous silicon.

The recesses 101, 102, 103 may, as described above, further comprise substances for a PCR, for example in freeze-dried form, in particular polymerases, nucleotides, salts and also PCR enhancers. In addition, you can additional primers required for the PCR, for example for a concentration between 0.1 and 1.5 micromolar, and further capture primers, for example for a concentration between 0.001 and 1 micromolar, in the recesses 101, 102, 103 be upstream, the latter to the PCR to support and in particular to allow a PCR in the liquid phase. These substances and primers can be wholly or partially in dehydrated, especially in freeze-dried form in the recesses 101, 102, 103, to be re-hydrated and thus activated when filling a liquid sample to be sequenced with nucleotide strands. In order to prevent carryover of reagents from the recesses, the in the

Recesses stored substances should be superimposed with polyethylene glycol (PEG). Such an application of PEG in particular enables a delayed and temperature-induced absorption of the substances into a liquid, in particular into the sample liquid.

FIG. 1 also shows schematically that the reaction carrier 100 can preferably be releasably connected to the device 200, for example via latching hooks 141, 142. Furthermore, it is shown schematically that the

Device 200 has a region 210 for a duplication and preferably dilution of the nucleotide strands to be sequenced. In particular, this region 210 can be embodied as a chamber 210 in the device 200, which is fluidically connected to the reaction carrier via a valve 211.

As shown in FIG. 2 a, the magnetic particles 110 can be fixed in the depressions 102 by a magnet 300, wherein the magnet 300 can be arranged, for example, in a second region 105 adjoining the reaction region 101. In FIG. 2b, by way of example, two particles 110 with immobilized capture molecules 121 are shown, to which sequenceable nucleotide strands 400 are bound. Sequencing primers 401 are already bound to the immobilized nucleotide strands 400 for sequencing by means of a sequencing-by-synthesis method on one of the two particles 110.

FIG. 3 shows a flowchart for an embodiment of the invention

The method 500 according to the invention for determining a nucleotide sequence which can be used, for example, with the exemplary embodiments described above Inventive Reaction carrier 100 and the inventive

Device 200 can be performed.

In a first step 501 of the method 500, the first capture primer 121 of the first recess 101 and second capture primer 122 are immobilized in the second recess 102 of the reaction support 100. If available, further capture primers can be immobilized in further recesses of the reaction support. As described above, this can preferably take place via a binding of the capture primers 121, 122, 123 to a mobile phase in the form of magnetic particles 110, which can be held in the recesses 101, 102, 103 of the reaction carrier 100 by means of a magnet 300. The binding of the capture primers 121, 122, 123 to the solid phase can be carried out, for example, via silanes, for example APTES, to which linkers are attached, for example 1,4-phenylene diisothiocyanate (PDITC), glutaraldehyde, s-SIAB, s-MBS, s-silane. GMBS, s-MBP. Also a direct one

Immobilization using poly (C, T) -tagged nucleic acids in combination with UV-initiated immobilization is possible (Y. Sun, R. Dhumpa, D. Bang, J. Hogberg, K. Handberg, A. Wolff, "A lab -on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR ", Lab Chip, vol. 11, pp. 1457-1463, 2011).

In a second step 502, nucleotide strands 400 to be sequenced are bound to the capture primers, preferably as part of a solid-phase PCR. Preferably, the recesses are during binding of the

Nucleotide strands 400 are closed to the capture primer, in particular by covering with a liquid, such as, for example, with an oil layer or a silicone layer, in order to ensure a contamination-free bond as possible. Before that, the nucleotide strands to be sequenced may have been amplified in an optional step, in particular by performing a PCR. This has the advantage that depending on the number of

Recesses a sufficient amount of nucleotide strands for the

Performing the second step 502 is provided.

Subsequently, a rinse of the particles 110 with a wash buffer, for example with a phosphate buffered saline solution (PBS for short), in a third Step 503, to optionally remove reaction components of the PCR. The bound to the catcher primers 121, 122, 123

Nucleotide strands may have two hydrogen-bonded sequences of nucleotides that are complementary to each other, and one end of a sequence is each bound to a capture primer or is the extension thereof. In other words, each are two mutually complementary nucleotide strands, one of the two nucleotide strands being directly bound to the capture primer. To prepare for the sequencing, the nucleotide strand not directly bound to the primer can be removed by dissolving the hydrogen bonds, for example using a sodium hydroxide solution. The caustic soda can be used both for the removal of these nucleotide strands and for the flushing described above. The caustic soda can a

Concentration of 0.1 molar and have other substances such as detergents or salts. Subsequently, it is possible to wash with tris- (hydroxymethyl) -aminomethane hydrochloride, tris-HCl for short, or saline solution, in order in particular to neutralize the sodium hydroxide solution.

In a fourth step 504, the sequence of the bound nucleotide strands is determined, in particular via a sequencing-by-synthesis method, which is described in US Pat

The following is exemplified. The fourth step 504 may preferably comprise the following steps five to ten.

In a fifth step 505, the sequencing primer 401 is added to the bound first nucleotide strands, wherein the sequencing primer 401 is designed to bind to a free end of the first nucleotide strands 400, as shown in Figure 2b. In a sixth step 506, nucleotides of one of a plurality of predetermined types are added to the bound first nucleotide strands, the added nucleotides having a label, preferably a fluorophore, and a polymerase terminator. In particular, the prescribed varieties are adenosine triphosphate, guanosine triphosphate, cytidine triphosphate and

Thymidine triphosphate. Examples of suitable fluorophores are

Cyanine dyes such as Cy3, or Cy5 or Atto equivalents. For example, the polymerase terminator is an esterification of the 3 ' Hydroxyl group of a nucleotide. The addition is carried out with the addition of polymerase, so that each binding of one of the added nucleotides to the bound nucleotide strands takes place when a complementarity of the added nucleotide with the bound in relation to the

Sequencing primer nearest nucleotide is present without complementary nucleotide of a respective bound first nucleotide strand. The addition of the polymerase can be carried out together or before the addition of the nucleotides. In a seventh step 507, unbound nucleotides are removed, preferably via a rinse of the solid phase, for example with PBS, before, in an eighth step 508, a detection of the bound nucleotides takes place via detection of the label. For optical detection, a conventional optical system consisting of excitation light, excitation filter, detection filter and detector can be used.

If the sequence of the bound nucleotides via the detection of the labels is not yet determined to a desired length, removal of the label and the polymerase terminator takes place in a ninth 509 step, for example via rinsing of the bound first nucleotide strands with Na 2 PdCl 4 / P (PhSO 3Na) 3 as a buffer when using 3'-0-allyl groups as a polymerase terminator. This will be tied to the first

Nucleotide strands generated free 3'-hydroxy ends, which allows incorporation of the next complementary nucleotide. Subsequently, in a tenth step 510, a new addition of polymerase and of nucleotides of one of the other prescribed varieties with label and

Polymerase terminator and a repetition of the above steps seven 507 to ten 510 until the sequence of the bound nucleotides to the desired or predetermined length on the detection of the label in one of the eighth steps 508 is determined.

Claims

claims

A reaction carrier (100) for a microfluidic device (200) for determining a nucleotide sequence comprising at least a first recess (101) and a second recess (102), wherein in the first recess (101) first capture primer (121) and in the second capture primer (122) are immobilized on a solid phase (110), wherein the first capture primers (121) are different from the second capture primers (122).

The reaction carrier (100) of claim 1, wherein the solid phase (110) comprises magnetic particles (110).

3. reaction carrier (100) according to any one of the preceding claims, wherein the

Solid phase (110) at least part of a wall (131) or a bottom (132) of the first and / or the second recess (101, 102).

4. reaction carrier (100) according to any one of the preceding claims, wherein the

Solid phase (110) comprises porous material, wherein the first and / or second capture primers (121, 122) are immobilized in pores of the porous material.

5. reaction carrier (100) according to any one of the preceding claims, wherein in

at least one of the recesses (101, 102, 103) further primers (121, 122, 123) for the determination of the nucleotide sequence in unbound form.

6. reaction carrier (100) according to any one of the preceding claims, wherein in

at least one of the recesses (101, 102, 103) further primers (121, 122, 123) and / or reagents for carrying out an amplification reaction,

in particular a polymerase chain reaction, in particular in dried form.

7. IVlikrofluidische device (200) with a reaction carrier (100) according to any one of the preceding claims, wherein the reaction carrier (100) in the microfluidic device (200) is arranged.

The IV microfluidic device (200) of claim 7, wherein the device (200) comprises a region (210) for pre-amplification and, preferably, dilution of nucleotide strands (400) to be sequenced.

The microfluidic device (200) according to claim 7 or 8, wherein the microfluidic device (200) has a region (105) adjoining the reaction support (100), wherein the region (105) is designed for receiving a magnet (300) in that a magnetic field of the magnet (300) for fixing a magnetic or magnetizable solid phase (110) in the recesses (101, 102, 103) can extend into one or more of the recesses (101, 102, 103).

10. Method (500) for determining a nucleotide sequence with a reaction carrier (100) for a microfluidic device (200), comprising the steps:

Immobilizing (501) first capture primers (121) in a first

Recess (101) and second capture primers (122) in a second recess (122) on a solid phase (110) of the reaction support (100), wherein the first capture primers (121) are different from the second capture primers (122).

Binding (502) nucleotide strands (400) to the first and / or second capture primers (121, 122).

Determining (504) a sequence of bound nucleotide strands (400).

The method (500) of claim 10, wherein the nucleotide strands (400) are amplified via a polymerase chain reaction prior to binding to the capture primers (121, 122).

The method (500) of claim 10 or 11, wherein the immobilizing step (501) comprises binding of the capture primers (121, 122, 123) to a solid phase (110).

13. The method (500) according to any one of claims 10 to 12, wherein the binding of the

Capture primer (121, 122, 123) to the solid phase (110) before immobilization of the solid phase on the reaction support (100).

14. The method according to any one of claims 10 to 13, wherein during the binding (502) of the nucleotide strands (400) to the first and / or second capture primer (121, 122), a closure of the recesses (101, 102, 103), in particular through a cover with a non-polar liquid.