EP-4739798-A1 - SYSTEM, DEVICE, AND METHOD FOR DETECTING NUCLEIC ACIDS

Abstract

Provided herein is a system, a device that includes modules, a cartridge that includes components such as a microfluidic channels, and methods that relate to detecting nucleic acids and other constituents of biological samples. The present disclosure also relates to cartridges, devices, and methods for performing sample analysis, e.g., nucleic acid analysis such as PCR analysis of materials within the cartridges in a rapid manner.

Inventors

• BRADLEY, Sean Patrick
• YENT, Gregory D.
• MARTINEZ-TRIBOLET, Gabriel
• BAUER, Ruth Anne
• GARNER, Garrett Dwight
• BIEBER, Ryan Keith
• BOMAN, Jennifer Alese
• MALONE, Trevor

Assignees

• Sekisui Diagnostics, LLC

Dates

Publication Date

20260513

Application Date

20240705

Claims (20)

1. 1. A method of detecting one or a plurality of selected nucleic acids in a sample, the method comprising: a. providing a sample mixed with lysis solution to a sample introduction cup of a microfluidic cartridge wherein the sample introduction cup comprises a moveable cap providing a pressure tight seal and is fluidically connected to a pneumatic channel and port via a first opening and a sample introduction microchannel via a second opening wherein the sample introduction microchannel connects to a valve configured to selectively isolate the sample introduction cup and sample introduction microchannel, and wherein the pneumatic channel is fluidically connected to a sample conveyance pressure port on a top surface of the cartridge; b. closing the cap of the sample introduction cup; c. introducing pressure to the sample conveyance pressure port to introduce pressure above the level of the fluid in the sample introduction cup; d. actuating the valve to fluidically connect the sample introduction cup to the sample introduction microchannel, which is fluidically connected to a mechanical lysis chamber, wherein a plurality of magnetizeable beads are disposed in said mechanical lysis chamber; e. conveying the sample mixed with lysis solution to the mechanical lysis chamber; f. actuating the valve to fluidically disconnect the mechanical lysis chamber from the sample introduction cup; g. actuating at least two rotatable members comprising magnets to move the magnetizeable beads in the mechanical lysis chamber wherein the sample is converted to lysed cell constituents comprising nucleic acids; h. actuating the valve to fluidically connect the lysis chamber with a extraction chamber which comprises a reversibly binding medium; i. conveying the lysed cell constituents into the extraction chamber wherein the nucleic acids reversibly bind to the reversibly binding medium; j. actuating the valve to fluidicially connect a wash solution reservoir which comprises wash solution to an introduction cup of the extraction chamber, and further fluidically connects an output port of the extraction chamber to a waste reservoir; k. conveying wash solution to the extraction chamber to convey the lysed cell constituents not reversibly bound to the reversibly binding medium into the waste reservoir while retaining substantially all of the nucleic acids bound to the reversibly binding medium; l. actuating the valve to fluidicially connect an elution solution reservoir, which comprises elution solution, to an input port of the extraction chamber, and to further fluidically connect an output port of the extraction chamber to one or a plurality of a PCR mastermix reagent zones, which comprise PCR mastermix reagents; m. conveying elution solution to the extraction chamber to elute and convey nucleic acid eluted from the reversibly binding medium into the one or a plurality of PCR mastermix zones forming nucleic acid PCR admixture; n. conveying the nucleic acid PCR admixture into a mixing channel forming a mixed nucleic acid PCR admixture; o. actuating the valve to fluidically connect the mixing channel to two or a plurality of PCR chambers and a waste reservoir wherein each PCR chamber comprises PCR reagents comprising one or more polymerases, control PCR reagents and selected PCR reagents for a selected target, wherein the control and selected PCR reagents comprise fluorescently labeled primers and or fluorescently labeled probes; p. conveying the mixed nucleic acid PCR admixture into the two or a plurality of PCR chambers, wherein each of the PCR chambers is filled with mixed nucleic acid PCR admixture comprising the PCR reagents, and wherein any excess mixed nucleic PCR admixture which is not required to fill the two or a plurality of PCR chambers is conveyed into the waste reservoir; q. actuating the valve to fluidically isolate the two or a plurality of PCR chambers; r. contacting a selected region of the microfluidic cartridge with a first heating zone; s. modulating the selected region of the microfluidic cartridge to one or a plurality of temperatures; t. contacting the selected region of the microfluidic cartridge with a second heating zone; u. modulating the selected region of microfluidic cartridge to one or a plurality of temperatures; v. presenting an excitation light source to the two or a plurality of PCR chambers wherein a labeled control primer or probe emits control fluorescence and a labeled selected primer or probe emits selected fluorescence; w. measuring one or a plurality of selected photophysical properties from the emitted control fluorescence and selected fluorescence from each PCR chamber; repeating steps (r) through (v) for a selected number of instances, wherein the microfluidic cartridge comprises a sample introduction cup, one or a plurality of valves, one or a plurality of storage elements (each of which can optionally independently further comprise an elution solution, PCR mastermix reagents, and/or a control sample), a mechanical lysis chamber, an extraction chamber, a waste reservoir, a mixing channel, and two to ten PCR chambers.
2. 2. The method of claim 1, wherein the valve is rotatable.
3. 3. The method of claim 1, wherein the microfluidic cartridge comprises one valve.
4. 4. The method of claim 1, wherein the sample introduction microchannel comprises a control reagent zone comprising one or more control reagents.
5. 5. The method of claim 1, wherein the lysis solution, elution solution, or both comprise one or more control reagents.
6. 6. The method of any of claims 4 or 5, wherein the one or more control reagents comprises a nucleic acid having a known sequence.
7. 7. The method of claim 6, wherein the control sample further comprises carrier RNA (cRNA).
8. 8. The method of claim 7, wherein the cRNA is greater than 200 nt (nucleotides) in length.
9. 9. The method of claim 7, wherein cRNA is polyA.
10. 10. The method of claim 6, wherein the nucleic acid having a known sequence is MS2 DNA (SEQ ID NO: 1).
11. 11. The method of claim 4, wherein the one or more control reagents are lyophilized.
12. 12. The method of claim 1, wherein step (c) introducing pressure to the sample conveyance pressure port to introduce pressure into the sample introduction cup, further comprises: (i) introducing pressure into a first reservoir of the sample introduction cup through a vertically aligned channel surrounded by a wall disposed within the interior of the sample introduction cap wherein at least a portion of the vertically aligned channel wall does not extend throughout the entirety of the first reservoir of the sample introduction cup and the top surface of the sample mixed with lysis solution is above the top of the vertically aligned channel, wherein pressure is increased in the volume defined by the top of the sample surface and the interior surface of the cap; (ii) stopping the introduction of pressure into said first reservoir and allowing the pressure presented to the first reservoir to return to atmospheric pressure, whereby any fluid in the first reservoir positioned above the top of the vertically aligned channel is conveyed through the vertically aligned channel into the lysis reservoir; (iii) introducing pressure to the sample conveyance pressure port to the sample holder cup.
13. 13. The method of claim 1, wherein each of the at least two rotatable members independently comprises magnets on one or both ends.
14. 14. The method of claim 1, wherein the reversibly binding medium is a silica medium.
15. 15. The method of claim 14, wherein the silica medium is a porous silica membrane.
16. 16. The method of claim 1, wherein conveying the sample mixed with lysis solution into the mechanical lysis chamber is performed by introduction of pressure to a pneumatic pump port.
17. 17. The method of claim 1, wherein conveying the lysed cell constituents into the extraction chamber is performed by introduction of pressure to a post-sample air pump port while the valve is actuated to fluidically disconnect the sample introduction cup from the mechanical lysis chamber.
18. 18. The method of claim 1, wherein conveying wash solution to the extraction chamber is performed by introduction of pressure to a wash solution reservoir pump port.
19. 19. The method of claim 1, wherein conveying elution solution to the extraction chamber is performed by introduction of pressure via an external disc pump in contact with an elution solution reservoir pump port.
20. 20. The method of claim 1, wherein conveying the nucleic acid PCR admixture into the mixing chamber is performed by introduction of pressure to an elution solution reservoir pump port.

Description

SYSTEM, DEVICE, AND METHOD FOR DETECTING NUCLEIC ACIDS RELATED APPLICATIONS This disclosure claims priority to U.S. Provisional Application No. 63/525,348, filed July 6, 2023, the contents of which are herein incorporated by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 1, 2024, is named U3415-00101 jSEQ_ID_LISTING.xml and is 9,867 bytes in size. FIELD OF THE INVENTION [0001] This disclosure relates to a system, a device that includes modules, a cartridge that includes components such as a microfluidic channels, and methods that relate to detecting nucleic acids and other constituents of biological samples. The present disclosure also relates to cartridges, devices, and methods for performing sample analysis, e.g., nucleic acid analysis such as PCR analysis of materials within the cartridges in a rapid manner. BACKGROUND [0002] Polymerase Chain Reaction (PCR) is a facile method involved in the detection of selected nucleic acids in a sample suspected of or having the selected nucleic acids by amplifying nucleic acids. Biological samples (e.g., saliva, buccal swab, nasal aspirate, blood, or other bodily fluids) comprise nucleic acids and many sources of PCR inhibitors. As such, PCR analysis of biological samples requires removal of such inhibitors to generate a nucleic acid sample of sufficient quality to perform the intended analysis. Manual sample preparation methods may often be time-consuming. [0003] Integrated microfluidic handling systems that provide control over limited sample volumes are useful in miniaturizing PCR tests in a time-efficient manner. Microfluidic devices include components such as channels, valves, pumps, flow sensors, mixing chambers and optical detectors. Examples of these components and systems may be found in U.S. Pat. Nos. 5,932,100; 5,922,210; 6,387,290; 5,747,349; 5,748,827; 5,726,751; 5,716,852; 5,974,867; 6,007,775; 5,972,710; 5,971,158; 5,948,684; 6,171,865; 7,648,835; 7,745,207 6,870,185; 8,672,532; and 8,894,946. SUMMARY OF THE INVENTION [0004] In one aspect, this disclosure relates to methods of preparing samples and detecting constituents from biological samples, for example, methods of loading metered amounts of sample into an analyzer, methods of lysing samples, and methods of detecting constituents, e.g., nucleic acids, polypeptides, peptides and/or post-translational modifications of polypeptides or peptides, from lysed samples. The present disclosure also relates, in some aspects, to detecting nucleic acids. In some aspects, this disclosure features methods of detecting one or a plurality of selected nucleic acids. [0005] In some aspects, the method comprises the steps of (a) providing a sample mixed with lysis solution to an introduction element of a microfluidic cartridge and (b) closing the cap of the introduction element, e.g., a cup, vial, tube, cylinder. The sample introduction element comprises a moveable cap providing a pressure tight seal. In some aspects, the sample introduction element is fluidically connected to a waste reservoir via a first opening and a sample introduction microchannel via a second opening, directly, or indirectly (e.g., through a valve) to other components of a system for purifying and/or detecting biological samples. In some aspects, the sample introduction microchannel connects to a valve configured to selectively isolate the sample introduction element and sample introduction microchannel. [0006] In one aspect, an introduction element for introducing a sample to a microfluidic cartridge includes an opening through the introduction element defined by annular ring. In one aspect, a chimney is positioned within the opening. The chimney may include an aperture extending through the chimney. The chimney aperture has an internal cross-sectional area that is less than an internal cross-sectional area of the opening. For example, in some aspects a cross-sectional area of the aperture is, from 5 to 65 % of a cross-sectional area of the opening. In some aspects, the cross-sectional area of the aperture is from 5 to 20 %, or from 5 to 15%, of a cross-sectional area of the opening. In some aspects, the cross-sectional area of the aperture is about 6.5% of a cross-sectional area of the opening. The chimney is, in some aspects, a hollow cylinder defined by the aperture and the opening. The chimney may extend beyond the annular ring defining the opening at both ends. The top of the chimney is submerged when a sample with a volume greater than a selected volume is presented to the sample introduction cup. In some aspects, the selected volume is about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, or 400 microliters, or any volume between any of the aforementioned volumes. Upon venting the sample introduction cup, bubbles may form which can be r