US-12618106-B2 - Methods and systems for extraction, processing, and detection of nucleic acids

Abstract

A removable cartridge to be used in a system for extracting and detecting nucleic acids from heterogeneous samples includes a plurality of reservoirs defining at least a first wash buffer reservoir for holding a first wash buffer and a microfluidic assembly configured to attach to the plurality of reservoirs. The microfluidic assembly includes at least one sample reservoir and a nucleic acid extraction matrix in fluid communication to an automated sample preparation (ASP) reservoir through a first flow channel defined by the microfluidic assembly. An assay chamber is in fluid communication with a third flow channel and with the waste reservoir through a fourth flow channel such that a labeled nucleic acid-containing sample flows through the assay chamber and then to the waste reservoir, wherein vibration-driven mixing agitates fluids while present in the assay chamber. Finally, a nucleic acid-detecting microarray module is positioned in the assay chamber.

Inventors

• Charles A. Lissandrello
• Aditi R. NAIK
• Diana J. LEWIS
• Erin Rosenberger
• Joseph Neil URBAN
• Jason Fiering
• Caleb R. Bell
• Cait Ni Chleirigh
• Ernest Kim

Assignees

• THE CHARLES STARK DRAPER LABORATORY, INC.

Dates

Publication Date

20260505

Application Date

20230501

Claims (20)

1. 1 . A cartridge for extracting and detecting nucleic acids from heterogeneous samples comprising: a plurality of reservoirs defining at least a first wash buffer reservoir configured to hold a first wash buffer; a microfluidic assembly, configured to attach to the plurality of reservoirs, comprising: at least one sample reservoir positioned in a first input defined by the microfluidic assembly; a nucleic acid extraction matrix positioned in the microfluidic assembly and in fluid communication to an automated sample preparation (ASP) reservoir through a first flow channel defined by the microfluidic assembly, a waste reservoir defined within the microfluidic assembly; a valve assembly configured to provide a first setting that permits flow from the first wash buffer reservoir through the nucleic acid extraction matrix and then to the waste reservoir and a second setting that permits collection of a nucleic acid-containing sample; an assay chamber in fluid communication with a third flow channel and with the waste reservoir through a fourth flow channel, and configured to allow a labeled nucleic acid-containing sample flow through the assay chamber and then to the waste reservoir, wherein a vibration motor is proximate to an outer surface of a first side of the assay chamber, wherein the vibration motor provides vibration-driven mixing, and the assay chamber comprises air-filled cavities inside the assay chamber contacting an inner surface of the first side of the assay chamber, the vibration motor and the air-filled cavities configured to promote microfluidic mixing of fluid in the assay chamber; and a nucleic acid-detecting microarray module positioned in the assay chamber.
2. 2 . The cartridge of claim 1 , wherein the plurality of reservoirs further includes an elution reservoir for holding an elution buffer.
3. 3 . The cartridge of claim 2 , wherein the nucleic acid extraction matrix is in fluid communication with the elution reservoir through a second flow channel defined by the microfluidic assembly and with the first wash buffer reservoir.
4. 4 . The cartridge of claim 1 , wherein the microfluidic assembly further includes an output collection vessel positioned in the microfluidic assembly.
5. 5 . The cartridge of claim 4 , wherein the first setting permits flow from the first wash buffer reservoir through the nucleic acid extraction matrix and then to the waste reservoir and the second setting that permits flow from an elution reservoir to the output collection vessel.
6. 6 . The cartridge of claim 1 , wherein the microfluidic assembly further includes an assay input vessel positioned in the microfluidic assembly.
7. 7 . The cartridge of claim 6 , wherein the assay chamber is in fluid communication with the assay input vessel through the third flow channel and with the waste reservoir through the fourth flow channel such that the nucleic acid-containing sample flows from the assay input vessel through the assay chamber and then to the waste reservoir.
8. 8 . The cartridge of claim 1 , wherein the third flow channel includes a first serpentine flow channel section and the fourth flow channel includes a second serpentine flow channel section.
9. 9 . The cartridge of claim 1 , wherein the plurality of reservoirs further including a second wash buffer chamber for holding a second wash buffer and a third wash buffer chamber holding a third wash buffer.
10. 10 . The cartridge of claim 9 , wherein the microfluidic assembly defines a fifth flow channel that is in fluid communication with the second wash buffer chamber and the third flow channel.
11. 11 . The cartridge of claim 10 , wherein the microfluidic assembly defines a sixth flow channel that is in fluid communication with the second wash buffer chamber and the third flow channel.
12. 12 . The cartridge of claim 1 , further comprising a closed-loop thermal control system that maintains steady temperature in the assay chamber.
13. 13 . The cartridge of claim 12 , wherein the closed-loop thermal control system includes a heater for heating the assay chamber and a temperature probe for measuring temperature of the assay chamber.
14. 14 . The cartridge of claim 1 , wherein the nucleic acid extraction matrix is incorporated in a cylindrical “spin column”.
15. 15 . The cartridge of claim 14 , wherein the spin column is in fluid communication with microchannels at its end faces.
16. 16 . The cartridge of claim 14 , wherein the spin column is fabricated by removing excess length from a spin column designed for centrifugation use.
17. 17 . The cartridge of claim 1 , wherein the microfluidic assembly is composed of one or more polymers.
18. 18 . The cartridge of claim 17 , wherein the microfluidic assembly is composed of a component selected from the group consisting of polycarbonate, acrylic, acrylonitrile butadiene styrene, polyethylene terephthalate, nylon, polypropylene, polystyrene, and combinations thereof.
19. 19 . The cartridge of claim 17 , wherein the microfluidic assembly includes multiple polymer layers that define portions of the microfluidic assembly.
20. 20 . The cartridge of claim 1 wherein a removable heater is mounted to the nucleic acid extraction matrix during use and demounted for disposal of the cartridge.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. provisional application Ser. No. 63/367,516 filed Jul. 1, 2022, the disclosure of which is hereby incorporated in its entirety by reference herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Contract No. N66001-21-C-4048, awarded by the U.S. Defense Advanced Research Projects Agency (DARPA). The Government has certain rights in the invention. TECHNICAL FIELD In at least one aspect, the present invention is related to methods and equipment for extracting and detecting nucleic acids. BACKGROUND Nucleic acid extraction from biological samples is typically performed in a laboratory environment using specialized equipment and requiring trained personnel. The often-low abundance of nucleic acid targets in biological samples forces constraints on detection systems including the need for large sample volumes, long incubation durations, and highly-sensitive assays. In addition, system performance may also be limited by the time required for the diffusive motion of targets to their respective probes if there are no means provided to improve transport beyond the diffusion limit. Sensitive and multiplexed detection of nucleic acid targets from biological samples is also a challenging problem that is typically addressed using specialized equipment in a laboratory setting. The process involves many steps including sample lysis, nucleic acid extraction, nucleic acid detection, and readout. Each of these steps is often achieved using separate pieces of equipment, specialized processing kits, or a sequence of manual steps which require user intervention. Accordingly, there is a need for improved methods of extracting and detecting nucleic acids. SUMMARY In at least one aspect, a removable cartridge to be used in a system for extracting and detecting nucleic acids from heterogeneous samples is provided. The removable cartridge includes a plurality of reservoirs defining at least a first wash buffer reservoir for holding a first wash buffer and a microfluidic assembly configured to attach to the plurality of reservoirs. The microfluidic assembly includes at least one sample reservoir positioned in a first input defined by the microfluidic assembly and a nucleic acid extraction matrix positioned in the microfluidic assembly and in fluid communication to an automated sample preparation (ASP) reservoir through a first flow channel defined by the microfluidic assembly. The microfluidic assembly also includes a waste reservoir defined within the microfluidic assembly and a fluid switching assembly configured to provide a first setting that permits flow from the first wash buffer reservoir through the nucleic acid extraction matrix and then to the waste reservoir and a second setting that permits collection of a nucleic acid-containing sample. An assay chamber is in fluid communication with a third flow channel and with the waste reservoir through a fourth flow channel such that a labeled nucleic acid-containing sample flows through the assay chamber and then to the waste reservoir, wherein vibration-driven mixing agitates fluids while present in the assay chamber. Finally, a nucleic acid-detecting microarray module is positioned in the assay chamber. In another aspect, a mixing chamber for a removable cartridge used in a system for extracting and detecting nucleic acids from heterogeneous samples is provided. The mixing chamber includes an assay chamber having a predetermined thickness and a first side having an outer surface and an inner surface. Characteristically, the assay chamber is configured to receive a fluid therein. A vibration motor is affixed to or proximate to the outer surface of the assay chamber. A plurality of air-filled cavities are in contact with both the outer surface wherein the vibration motor is affixed and the fluid being mixed. In another aspect, a Massively Multiplexed Detection (MMD) Device is provided. The MMD Device is capable of being a fieldable, battery-powered, shelf-stable system which leverages gene-editing technologies and combines novel mechanical elements to achieve highly-multiplexed and sensitive detection of up to 1000 nucleic acid targets. The system is preferably paired with a custom disposable cartridge which contains the assay array and all reagents needed for processing. The system and cartridge together provide an all-in-one sample-to-answer solution for sample lysis, nucleic acid extraction, nucleic acid detection, and readout. In another aspect, a nucleic acid extraction module includes three or more integrated reagent storage reservoirs (lysis buffer, wash buffer, and elution buffer), three or more passive umbrella valves, one or more pinch valves, a pump or pressure source, one or more motorized rotary valves, a heater, a column for nucleic acid extraction, several microfluidic channels, and connections for sample and reage