Microfluidic Sequencing Device for Multiplying and Separating Molecular Chains, and Method for Separating Molecular Chains Obtained from an Amplification Reaction

Abstract

A microfluidic sequencing device for multiplying and separating molecular chains, and which is designed to receive biochemical material, includes at least one supply opening for supplying biochemical material into the sequencing device. The sequencing device additionally has at least one microfluidic separation unit, which has at least one separation channel with at least one amplification cavity for multiplying molecular chains supplied via the supply opening as the biochemical material and with at least one separating unit which is connected or can be connected to the amplification cavity microfluidically and includes a multi-porous material. The separating unit is designed to separate nucleic acids from other macromolecular components and/or to separate nucleic acids. Furthermore, the sequencing device has at least one discharge opening for discharging nucleic acids separated in the separation unit as biochemical material out of the sequencing device.

Claims

1. A microfluidic sequencing device for reproducing and separating molecular chains, the sequencing device configured to accommodate biochemical material, the sequencing device comprising: a supply opening configured for supplying biochemical material into the sequencing device; at least one microfluidic separation-procedure unit comprising at least one separation-procedure channel comprising (i) at least one amplification cavity configured to reproduce molecular chains supplied via the supply opening as biochemical material, and (ii) at least one separation unit microfluidically connectable or connected to the at least one amplification cavity and including a multipore material, the at least one separation unit configured to separate nucleic acids from further macromolecular constituents and/or to resolve nucleic acids; and a discharge opening configured for discharging nucleic acids resolved in the at least one separation-procedure unit from the sequencing device as biochemical material.

2. The sequencing device as claimed in claim 1, wherein the at least one separation unit comprises at least one of the following: porous silicon, polycarbonate filter membranes, fabric-type polymer membranes, nanoporous metal oxide, an array of microposts, an acrylamide gel, and an agarose gel.

3. The sequencing device as claimed in claim 1, wherein the at least one separation unit comprises at least two multipore layers of different porosity.

4. The sequencing device as claimed in claim 1, wherein the at least one separation unit has a width which substantially corresponds to a width of the at least one amplification cavity.

5. The sequencing device as claimed in claim 1, wherein the at least one amplification cavity is connectable or connected to the at least one separation unit by a wall having a bottleneck.

6. The sequencing device as claimed in claim 1, wherein the supply opening and/or the discharge opening is formed as a constriction of the at least one separation-procedure channel.

7. The sequencing device as claimed in claim 1, further comprising: a field-development unit configured to generate an electric field across the at least one separation unit in order to convey a molecular chain through the at least one separation unit by electrophoresis.

8. The sequencing device as claimed in claim 1, wherein: at least one reaction component and at least one artificial DNA nucleotide and/or one chain-termination nucleotide is kept in reserve in the at least one amplification cavity; and/or the at least one amplification cavity is configured to accommodate a liquid for an amplification reaction.

9. The sequencing device as claimed in claim 1, wherein: the at least one amplification cavity of the at least one separation-procedure unit comprises at least two amplification cavities, and each of the at least two amplification cavities contains (a) at least one identical reaction component and (b) at least one different, artificial DNA nucleotide and/or at least one respectively different chain-termination nucleotide; and/or each of the at least two amplification cavities contains is designed to accommodate a liquid for an amplification reaction.

10. The sequencing device as claimed in claim 1, further comprising: a length-determination unit configured to determine a length of resolved nucleic acids as biochemical material.

11. A method for separating molecular chains obtained from an amplification reaction, using a microfluidic sequencing device that has (a) a supply opening configured for supplying biochemical material into the sequencing device; (b) at least one microfluidic separation-procedure unit including at least one separation-procedure channel that includes (i) at least one amplification cavity configured to reproduce molecular chains supplied via the supply opening as biochemical material, and (ii) at least one separation unit microfluidically connectable or connected to the at least one amplification cavity and including a multipore material, the at least one separation unit configured to separate nucleic acids from further macromolecular constituents and/or to resolve nucleic acids; and a discharge opening configured for discharging nucleic acids resolved in the at least one separation-procedure unit from the sequencing device as biochemical material, the method comprising: introducing a liquid for the amplification reaction into at least one amplification cavity of at least one separation-procedure channel of at least one microfluidic separation-procedure unit in order to prepare for an amplification reaction; conducting a sealing liquid across the supply opening and the discharge opening in order to seal the supply opening and the discharge opening; performing the amplification reaction in order to generate at least one molecular chain and terminating obtained molecular chains in order to generate chain-termination products; and applying a voltage between the supply opening and the discharge opening in order to fill the at least one separation unit and separating the at least one molecular chain generated in the separation unit in order to obtain chain-termination products;

12. The method as claimed in claim 11, further comprising: measuring a length of the chain-termination products in order to detect a sequence of the chain-termination products.

13. A method for producing the sequencing device as claimed in claim 1, the method comprising: forming the at least one separation unit of the multipore material; and connecting the at least one separation unit to the at least one amplification cavity in the at least one separation-procedure unit in order to produce the sequencing device.

14. A device which is configured to execute and/or control the steps of the method as claimed in claim 11 in corresponding units.

15. A computer program which is configured to execute and/or control a microfluidic sequencing device, which has (a) a supply opening configured for supplying biochemical material into the sequencing device; (b) at least one microfluidic separation-procedure unit including at least one separation-procedure channel that includes (i) at least one amplification cavity configured to reproduce molecular chains supplied via the supply opening as biochemical material, and (ii) at least one separation unit microfluidically connectable or connected to the at least one amplification cavity and including a multipore material, the at least one separation unit configured to separate nucleic acids from further macromolecular constituents and/or to resolve nucleic acids; and a discharge opening configured for discharging nucleic acids resolved in the at least one separation-procedure unit from the sequencing device as biochemical material, so as to execute program instructions stored in memory to operate the microfluidic sequencing device to separate molecular chains obtained from an amplification reaction by: introducing a liquid for an amplification reaction into at least one amplification cavity of at least one separation-procedure channel of at least one microfluidic separation-procedure unit in order to prepare for an amplification reaction; conducting a sealing liquid across the supply opening and the discharge opening in order to seal the supply opening and the discharge opening; performing an amplification reaction in order to generate at least one molecular chain and terminating obtained molecular chains in order to generate chain-termination products; and applying a voltage between the supply opening and the discharge opening in order to fill the at least one separation unit and separating the at least one molecular chain generated in the separation unit in order to obtain chain-termination products.

16. The method as claimed in claim 15, wherein the computer program is stored on a machine-readable storage medium.

17. The sequencing device as claimed in claim 2, wherein the polycarbonate filter membranes are track-etched membranes.

18. The sequencing device as claimed in claim 9, wherein the at least two amplification cavities includes at least four amplification cavities.

Description

[0044] Exemplary embodiments of the approach presented here are depicted in the drawings and more particularly elucidated in the following description, where:

[0045] FIGS. 1A to 1C show a schematic representation of a sequencing device according to various exemplary embodiments;

[0046] FIG. 2 shows a schematic representation of a sequencing device according to a further exemplary embodiment;

[0047] FIG. 3 shows a schematic representation of a sequencing device according to a further exemplary embodiment;

[0048] FIG. 4 shows a flow chart of a method for reproducing and separating molecular chains according to one exemplary embodiment;

[0049] FIG. 5 shows a flow chart of a method for producing a sequencing device according to one exemplary embodiment.

[0050] FIG. 6 shows a schematic representation of a device for executing or controlling the steps of a method for producing a sequencing device according to one exemplary embodiment.

[0051] FIG. 7 shows a schematic representation of a device for executing or controlling the steps of a method for reproducing and separating molecular chains according to one exemplary embodiment.

[0052] In the following description of favorable exemplary embodiments of the present invention, identical or similar reference signs are used for the elements which are depicted in the various figures and act in a similar manner, in order to dispense with a repeated description of said elements.

[0053] FIG. 1A shows a schematic representation of a cross-section of a microfluidic sequencing device 100 according to one exemplary embodiment. The sequencing device 100 is designed to accommodate biochemical material and to reproduce and separate molecular chains. The sequencing device 100 comprises at least one separation-procedure unit 101. The separation-procedure unit 101 comprises at least one separation-procedure channel 102. By way of example, this exemplary embodiment shows two such separation-procedure channels 102. The separation-procedure unit 101 comprises at least one supply opening 105, at least one microfluidic separation-procedure channel 102 comprising an amplification cavity 115 and a separation unit 120, and one discharge opening 125. The supply opening 105 is designed to supply biochemical material into the sequencing device 100. The at least one separation-procedure channel 102 is designed to microfluidically connect the amplification cavity 115 to the separation unit 120. The amplification cavity 115 is designed to reproduce molecular chains. The separation unit 120 consists of multipore material and is designed to separate nucleic acids from further macromolecular constituents and/or to resolve nucleic acids. The discharge opening 125 is designed to discharge biochemical material from the sequencing device 100.

[0054] According to one exemplary embodiment, biochemical material, especially a liquid for an amplification reaction, can be introduced into the amplification cavity 115 via the supply opening 105. Connected to the amplification cavity 115 is the separation unit 120, which opens into the discharge opening 125. Moreover, the separation-procedure unit 101 is, according to one exemplary embodiment, formed to be electrically contactable with a voltage source 135 at the supply opening 105 and at the discharge opening 125 via a field-development unit 130 in order to convey PCR products through the separation path by electrophoresis. Here, the field-development unit 130 can use either direct voltage or alternatively alternating voltage.

[0055] According to one exemplary embodiment, the separation unit 120 comprises porous silicon at least in part and/or polycarbonate filter membranes at least in part, especially track-etched membranes. Additionally or alternatively, the separation unit 120 comprises fabric-type polymer membranes at least in part and/or nanoporous metal oxide at least in part and/or an array of microposts at least in part and/or an acrylamide gel at least in part and/or an agarose gel at least in part. The porous character of the separation unit 120 increases the residence time in the separation unit 120, and this improves the differentiability of DNA fragments of different length.

[0056] According to one exemplary embodiment, the separation unit 120 has a width which substantially corresponds to the width of the amplification cavity 115.

[0057] According to one exemplary embodiment, the supply opening 105 is arranged at the entrance of the amplification cavity 115, in the flow direction of supplying biochemical material, especially the liquid for the amplification reaction. According to said exemplary embodiment, the discharge opening 125 is substantially formed in the width of the separation unit 120, at the exit of the separation unit 120, from which the biochemical material, especially the separated chain-termination products, is discharged.

[0058] FIG. 1B shows a schematic representation of a cross-section of a microfluidic sequencing device 100 according to a further exemplary embodiment. Said exemplary embodiment differs from the preceding one only in the formation of the discharge opening. In said exemplary embodiment, the discharge opening 125b is formed as a constriction at the exit of the separation unit 120.

[0059] FIG. 1C shows a schematic representation of a cross-section of a microfluidic sequencing device 100 according to a further exemplary embodiment. Said exemplary embodiment differs from the two preceding ones only by a bottleneck 140 between the amplification cavity 115 and the separation unit 120. The discharge opening 125a corresponds to the formation depicted in FIG. 1A. According to one exemplary embodiment, the amplification cavity 115 is connectable or connected to the separation unit 120 by a wall having a bottleneck 140.

[0060] The amount of the biochemical material, especially an amount of DNA, present in the separation unit 120 during the separation process is limited so as to minimize mixed signals (when too many DNA fragments of different length are situated and/or overlap in the separation region) in a measurement.

[0061] FIG. 2 shows a schematic representation of a sequencing device 100 according to a further exemplary embodiment. Said exemplary embodiment differs from the preceding ones only by the formation of the separation unit 120. According to said exemplary embodiment, the separation unit 120 comprises at least two multipore layers 210, 220 of different porosity. By way of example, this representation shows a first multipore layer 210 and a second multipore layer 220, which have differing porosity. The series connection of layers 210, 220 of different porosity especially allows various separation steps, for example that macromolecular constituents are removed in a first layer 210, whereas the chain-termination products, especially nucleic acids (DNA fragments), are resolved in a second layer 220.

[0062] FIG. 3 shows a schematic representation of a sequencing device 100 according to a further exemplary embodiment. Said exemplary embodiment differs from the preceding ones only by the content of the amplification cavity 115 and by the number of the depicted separation-procedure channels in the separation-procedure channel apparatus 101. Moreover, the separation-procedure channel apparatus 101 consists here, by way of example, of two segments, which each comprise four separation-procedure channels 102 comprising the amplification cavity 115 and the separation unit 120, the segments of the separation-procedure channel apparatus 101 being shown here as counter-images (in a mirrored manner).

[0063] According to this exemplary embodiment, at least one reaction component and at least one artificial DNA nucleotide and/or one chain-termination nucleotide of the bases A, G, T, C is kept in reserve in each amplification cavity 115. Additionally or alternatively, the amplification cavity 115 is designed to accommodate a liquid for an amplification reaction.

[0064] According to one exemplary embodiment, the separation-procedure unit 101 comprises at least two amplification cavities 115, especially four amplification cavities 115. By way of example, what are shown per segment of the separation-procedure unit 101 are four amplification cavities 115 each. The amplification cavities 115 are designed to contain at least one identical reaction component each and at least one different, artificial DNA nucleotide each and/or at least one respectively different chain-termination nucleotide A, G, T, C. Additionally or alternatively, the amplification cavity 115 is designed to accommodate a liquid for an amplification reaction.

[0065] According to the exemplary embodiment shown here, two different DNA sequences can be sequenced in the sequencing device 100, especially in each segment of the separation-procedure unit 101 one. To this end, the four amplification cavities 115 in each of the two segments of the separation-procedure unit 101 comprise for the generation of differently terminated chain-termination products 310 (especially terminated with A=adenine, G=guanine, T=thymine, C=cytosine). Each of the amplification cavities 115 is connected to one separation unit 120. By means of a field-development apparatus 130 and a voltage source 135, it is possible to generate an electric field across the separation units 120. In one operation, a dye is supplied to the liquid for the amplification reaction before or after the reaction, which dye allows a fluorometric detection of chain-termination products 310 obtained in the amplification reaction and resolved by the separation unit 120. It is thus possible to differentiate various chain-termination products 310 in a separation unit 120 in terms of their length.

[0066] FIG. 4 shows a flow chart of a method 400 for separating molecular chains obtained from an amplification reaction, according to one exemplary embodiment. The method 400 is executable using an already described microfluidic sequencing device 100 according to one of the exemplary embodiments, the method 400 comprising at least one introduction step 410, one conduction step 420, one performance step 430 and one application step 440.

[0067] In the introduction step 410, a liquid for an amplification reaction is introduced into the at least one amplification cavity in order to prepare for an amplification reaction. For example, a pressure difference is applied between the entrance of the amplification cavity and the exit of the separation unit for the introduction step in order to introduce the liquid for an amplification reaction into the separation-procedure channel. It is equally possible to utilize capillary forces for fluidic actuation, i.e., for the introduction of the liquid for an amplification reaction.

[0068] In the conduction step 420, a sealing liquid is conducted across the supply opening and the discharge opening in order to seal the supply opening and the discharge opening. In this connection, the discharge opening and the supply opening are flushed with a sealing liquid.

[0069] In the performance step 430, the conditions for an amplification reaction are provided and an amplification reaction is performed in order to generate at least one molecular chain. At the same time, the molecular chains obtained in the amplification reaction are terminated in order to generate chain-termination products. Termination is achieved by an artificial DNA nucleotide and/or a chain-termination nucleotide.

[0070] In the application step 440, a voltage is applied between the supply region and the discharge region in order to fill the at least one separation unit. Moreover, the at least one molecular chain generated is resolved in the separation unit in order to obtain chain-termination products. In addition, a running buffer can be admitted to the separation unit at its ends before the application of voltage. As a result, capillary filling of the separation unit occurs, and this improves the migration behavior of the chain-termination products upon application of an electric field.

[0071] In addition, FIG. 4 shows a flow chart of a method 450 for detecting chain-termination products. The method 450 comprises at least the steps of the method 400, namely an introduction step 410, a conduction step 420, a performance step 430 and an application step 440, and additionally a measurement step 460, which can take place at the same time as the application step 440.

[0072] In the measurement step 460, the length of the chain-termination products is measured in order to detect the sequence of the chain-termination products. For example, what is measured at the same time as the application step 440 is the current at the separation-procedure unit that is dependent on the amount of the chain-termination products and/or what is measured is the rate of passage of individual fragments of biochemical material. Additionally or alternatively, the resolved and previously stained chain-termination products are read optically.

[0073] FIG. 5 shows a flow chart of a method 500 for producing a sequencing device according to one presented exemplary embodiment. The method 500 comprises at least one formation step 510 and one connection step 520.

[0074] In the formation step 510, a separation unit composed of a multipore material is formed. The multipore separation unit can be formed by means of a coating method, especially a chemical vapor deposition (LPCVD). Additionally or alternatively, the separation unit can also be produced from porous silicon using a common method, such as in the case of wafer fabrication, and/or be hydrophilized or hydrophobized by means of vapor-coating, sputtering or silanization.

[0075] In the connection step, the separation unit is connected to an amplification cavity in a separation-procedure unit in order to produce the sequencing device.

[0076] FIG. 6 shows a schematic representation of a device 600 for executing and/or controlling the steps of a method for producing a sequencing device according to one exemplary embodiment.

[0077] The device 600 is configured to execute and/or control the steps of the method in corresponding units. The device comprises at least one formation unit 610 and one connection unit 620. The formation unit is designed to control and/or execute the step of forming a separation unit composed of multipore material and the connection unit 620 is designed to execute and/or control the step of connecting the separation unit to an amplification cavity in a separation-procedure unit.

[0078] FIG. 7 shows a schematic representation of a device 700 for executing and/or controlling the steps of a method 400 for reproducing and separating molecular chains and/or of a method 450 for detecting chain-termination products according to one exemplary embodiment. The device 700 is configured to execute and/or control the steps of the method in corresponding units. The device comprises at least one introduction unit 710, one conduction unit 720, one performance unit 730 and one application unit 740. The introduction unit is designed to introduce a liquid for an amplification reaction into the at least one amplification cavity in order to prepare for an amplification reaction. The conduction unit is designed to conduct a sealing liquid across the supply opening and the discharge opening in order to seal the supply opening and the discharge opening. The performance unit is designed to perform an amplification reaction in order to generate at least one molecular chain and to terminate the at least one molecular chain obtained in order to generate chain-termination products. The application unit is designed to apply a voltage between the supply opening and the discharge opening in order to fill the at least one separation unit and in order to separate the at least one molecular chain generated in the separation unit in order to obtain chain-termination products.

[0079] In addition, the device 700 can comprise a measurement unit which is designed to measure the length of the chain-termination products in order to detect the sequence of the chain-termination products.

[0080] If an exemplary embodiment comprises an “and/or” link between a first feature and a second feature, this is to be interpreted as meaning that the exemplary embodiment comprises both the first feature and the second feature according to one embodiment and either only the first feature or only the second feature according to a further embodiment.