# US 12,179,197 B2 · Microfluidic sequencing device for multiplying and separating molecular chains, and method for separating molecular chains obtained from an amplification reaction > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,179,197 B2 - Document ID: US12179197B2 - Status: Granted - Kind: B2 - Application number: 17/051,688 - Filed: 2019-05-02 - Published: 2021-08-05 - Granted: 2024-12-31 - Earliest claimed: 2018-05-08 - Assignees: Robert Bosch GmbH - Applicants: Robert Bosch GmbH - Inventors: Jochen Hoffmann - Web page: https://patentgrep.com/patent/US12179197B2 ## CPC - B01L 3/502753: {characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation} - B01L 3/50857: {using arrays or bundles of open capillaries for holding samples} - B01L 3/502707: {characterised by the manufacture of the container or its components} - B01L 3/50273: {characterised by the means or forces applied to move the fluids} - B01L 2300/0681: Filter - B01L 2400/0424: Dielectrophoretic forces - C12Q 1/6869: Methods for sequencing - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## 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 ### Claim 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, 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, and an array of microposts. ### Claim 2 (depends on 1) The sequencing device as claimed in claim 1, wherein the at least one separation unit comprises at least two multipore layers of different porosity. ### Claim 3 (depends on 1) 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. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 1) 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. ### Claim 8 (depends on 1) 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. ### Claim 9 (depends on 1) 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. ### Claim 10 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. ### Claim 11 (depends on 10) The method as claimed in claim 10, further comprising: measuring a length of the chain-termination products in order to detect a sequence of the chain-termination products. ### Claim 12 (depends on 10) A device which is configured to execute and/or control the steps of the method as claimed in claim 10 in corresponding units. ### Claim 13 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. ### Claim 14 (depends on 13) The method as claimed in claim 13, wherein the computer program is stored on a machine-readable storage medium. ### Claim 15 (depends on 1) The sequencing device as claimed in claim 1, wherein the polycarbonate filter membranes are track-etched membranes. ### Claim 16 (depends on 8) The sequencing device as claimed in claim 8, wherein the at least two amplification cavities includes at least four amplification cavities.