Methods and systems for sample processing and optical PCR

Abstract

A method and system for performing optical PCR. The method includes binding nucleic acids in a sample fluid to a solid phase substrate. The method further includes flowing the sample fluid in the fluid conduit to the capture site. The capture site may include a chamber. The method may further include applying a magnetic field to capture the solid phase substrate of the sample fluid flowing through the fluid conduit at a capture site. The method further includes flowing a wash buffer through the fluid conduit to remove impurities from the solid phase substrate. The method further includes flowing the immiscible fluid through the fluid conduit to remove residual sample fluid and / or wash buffer. The method further includes flowing an elution buffer through the fluid conduit to elute the nucleic acid from the solid phase substrate and performing optical PCR on the eluted nucleic acid.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 384,356, filed on November 18, 2022, which is hereby incorporated by reference in its entirety for all intents and purposes. Background of the Invention

[0003] The isolation of nucleic acids is an important step in many biochemical and diagnostic procedures. For example, nucleic acids need to be isolated from the complex mixtures in which they are often found before performing other studies and procedures (e.g., detection, cloning, sequencing, amplification, hybridization, cDNA synthesis, etc.). Methods for isolating nucleic acids from complex starting materials such as whole blood, serum, urine, and respiratory secretions include lysing the biological material in the presence of protein-inactivating conditions (e.g., protein-degrading enzymes) and then separating the nucleic acids from contaminants by methods including solvent precipitation, solid-phase extraction, and dialysis of nucleic acids. However, the presence of large amounts of cellular or other contaminating materials (e.g., proteins, carbohydrates, or salts) in such complex mixtures often hinders many of the reactions and techniques used in molecular biology. Thus, there is a need for improved methods for isolating and extracting nucleic acids from complex mixtures for use in the diagnosis of microbial infections, forensics, tissue and blood typing, detection of genetic variations, etc. Summary of the Invention

[0004] The present disclosure relates to methods and systems suitable for extracting nucleic acids from biological samples. In some embodiments, a system for performing PCR is provided. The system includes a cartridge that includes: a sample chamber that includes an inlet configured to receive a biological sample; a fluid conduit that communicates with the sample chamber, the fluid conduit including a capture site disposed along the length of the fluid conduit, the capture site having a cross-sectional area greater than the average cross-sectional area of the fluid conduit; a plurality of holes disposed in the body of the cartridge, wherein each of the holes is configured to receive one of a plurality of fluids, and wherein a portion of the plurality of holes is in fluid communication with the fluid conduit; and one or more magnetic field sources disposed adjacent to the capture site. The system includes a PCR device coupled to the cartridge. The PCR device includes an optical PCR cavity that includes a light absorption layer. In some embodiments, each of the plurality of holes is configured to receive a storage container that includes one of a plurality of fluids, wherein the storage container is configured to be pierced to release the fluid therein. In some embodiments, the plurality of fluids includes one or more of a lysis buffer, a wash buffer, an alcohol, an immiscible fluid, or an elution buffer. In some embodiments, at least one of the plurality of holes includes a solid phase substrate. In some embodiments, the plurality of holes includes: a first hole configured to receive a lysis buffer; a second hole configured to receive a first wash buffer; a third hole configured to receive a solid phase substrate; a fourth hole configured to receive an immiscible fluid; a fifth hole configured to receive a second wash buffer; and a sixth hole configured to receive an elution buffer. In some embodiments, the first hole and the third hole are in fluid communication with the sample chamber. In some embodiments, the second hole, the fourth hole, the fifth hole, and the sixth hole are in fluid communication with the fluid conduit. In some embodiments, the cartridge further includes a waste chamber. In some embodiments, the system includes a plurality of valves, wherein a first valve of the plurality of valves is configured to supply fluid from the sample chamber or the plurality of holes to the fluid conduit. In some embodiments, a second valve is configured to supply fluid from the fluid conduit to the PCR device. In some embodiments, the system further includes a pump in communication with the sample chamber, and wherein the pump is configured to provide pressure to move the sample fluid through the fluid conduit. In some embodiments, the capture site has a cross-sectional area that is at least 10% greater than the average cross-sectional area of the fluid conduit. In some embodiments, at least one of the one or more magnetic field sources is configured to move in a direction around the capture site of the fluid conduit. In some embodiments, the shape of the capture site is cylindrical, hexagonal, cuboid, or spherical. In some embodiments, the system includes a controller coupled to the one or more magnetic field sources, and wherein the controller is configured to change the position, intensity, or field of at least one of the one or more magnetic field sources. In some embodiments, the light absorption layer includes a material having a light absorption rate greater than 50%. In some embodiments, the system includes one or more heaters positioned adjacent to the capture site. In some embodiments, the one or more heaters are configured to heat the components in the capture site.In some embodiments, the optical PCR cavity is surrounded by an outer housing. In some embodiments, the outer housing includes one or more optical components, one or more heaters, and one or more temperature sensors. In some embodiments, the one or more heaters are configured to heat the sample during optical PCR.

[0005] In some embodiments, a method for performing PCR is provided. The method includes providing a system for processing a biological sample. The system includes a cartridge that includes: a sample chamber that includes an inlet configured to receive the biological sample; a fluid conduit that is in communication with the sample chamber, the fluid conduit including a capture site disposed along a length of the fluid conduit, the capture site having a cross-sectional area greater than an average cross-sectional area of the fluid conduit; and a plurality of holes disposed in a body of the cartridge, wherein each of the holes is configured to receive one of a plurality of fluids, and wherein a portion of the plurality of holes is in fluid communication with the fluid conduit; and one or more magnetic field sources disposed adjacent to the capture site. The method includes a PCR device in communication with the cartridge. The PCR device includes an optical PCR cavity that includes a light absorption layer configured for photothermal conversion. The method includes providing the biological sample to the inlet of the sample chamber. The method includes flowing a lysis buffer from a first one of the plurality of holes into the sample chamber to extract nucleic acids from the biological sample. The method includes flowing a solid phase substrate from a second one of the plurality of holes into the sample chamber to produce a sample fluid including the extracted nucleic acids bound to the solid phase substrate. The method includes flowing the sample fluid from the sample chamber into the fluid conduit. The method includes applying a magnetic field to capture the solid phase substrate in the sample fluid at the capture site of the fluid conduit. The method includes flowing a wash buffer through the fluid conduit to remove impurities from the solid phase substrate. The method includes flowing an immiscible fluid through the fluid conduit to remove residual sample fluid and / or wash buffer. The method includes flowing an elution buffer through the fluid conduit to elute nucleic acids from the solid phase substrate. The method includes transferring the eluted nucleic acids to the optical PCR cavity. The method includes performing PCR on the eluted nucleic acids in the optical PCR cavity. In some embodiments, the method includes mixing the lysis buffer and the biological sample to extract nucleic acids from the biological sample. In some embodiments, the method includes mixing the biological sample and the solid phase substrate to bind nucleic acids to the solid phase substrate. In some embodiments, the mixing includes performing bubble mixing using a pump. In some embodiments, flowing the immiscible fluid occurs after flowing the wash buffer. In some embodiments, the wash buffer includes an alcohol-based wash buffer. In some embodiments, the alcohol-based wash buffer includes ethanol or propanol. In some embodiments, the method does not include flowing air through the fluid conduit to dry the fluid conduit or the solid phase substrate. In some embodiments, the method includes applying a time-varying magnetic field to move the solid phase substrate in the fluid conduit, wherein the magnetic field is configured to maintain a sufficient force on the solid phase substrate to keep the solid phase substrate captured during at least one of: flowing the wash buffer through the fluid conduit; flowing the immiscible fluid through the fluid conduit; or flowing the elution buffer through the fluid conduit. In some embodiments, the capture site has a cross-sectional area that is at least 10% greater than the average cross-sectional area of the fluid conduit. In some embodiments, the capture site is characterized by a hexagonal shape. In some embodiments, the system further includes one or more heaters positioned adjacent to the capture site.In some embodiments, the method further includes heating the capture site using one or more heaters during at least one of: flowing sample fluid from a sample chamber into a fluid conduit; flowing a wash buffer through the fluid conduit; flowing an immiscible fluid through the fluid conduit; and / or flowing an elution buffer through the fluid conduit. In some embodiments, the method includes changing a magnetic field during at least one of: flowing a wash buffer through the fluid conduit; flowing an immiscible fluid through the fluid conduit; or flowing an elution buffer through the fluid conduit to move a solid-phase substrate within the capture site. In some embodiments, eluted nucleic acid from the solid-phase substrate is transferred into an elution buffer, wherein flowing the elution buffer includes flowing the eluted nucleic acid away from the capture site. In some embodiments, the immiscible fluid includes one or more of mineral oil, silicone oil, hexadecane, paraffin oil, fluorinated liquid, fluorinated oil, or a mixture thereof. In some embodiments, the solid-phase substrate includes a plurality of beads, paramagnetic beads, magnetic beads, glass beads, glass, or glass fiber. In some embodiments, the solid-phase substrate includes a coating that includes silica, ceramic, polymer, oligonucleotide, or a mixture thereof. In some embodiments, the solid-phase substrate includes a plurality of paramagnetic beads. In some embodiments, the fluid conduit includes a fluid capture region configured to receive the immiscible fluid. In some embodiments, a magnetic field is applied to a region of the capture site that is less than at least one dimension of the capture site. In some embodiments, flowing the elution buffer through the fluid conduit includes flowing the elution buffer in a direction opposite to the flow direction of the immiscible fluid.

[0006] Compared to conventional techniques, many benefits are achieved by the approach of the present invention. For example, embodiments of the present invention accelerate the overall extraction process, result in higher nucleic acid yields, and minimize interference with downstream assays for improved polymerase chain reaction (PCR) amplification. In some embodiments, flowing an immiscible fluid through the fluid conduit after one or more wash steps completely removes residual wash fluid, including residual contaminants, such that drying of the fluid conduit is unnecessary and it is possible to directly elute nucleic acid after this step. In some embodiments, the fluid conduit for nucleic acid extraction and separation includes a chamber for capturing a solid-phase substrate within the fluid conduit to increase contact between the fluid and the solid-phase substrate, thereby removing residual components (e.g., supernatant) from the solid-phase substrate. Additionally, embodiments of the present disclosure provide methods for sample processing and performing optical PCR. These and other embodiments of the present disclosure, along with many of the advantages and features of the present disclosure, are described in more detail below in conjunction with the accompanying text and corresponding drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The present disclosure will be readily understood by the following detailed description in conjunction with the accompanying drawings, wherein like reference numerals designate identical structural elements, and in the drawings:

[0008] Figure 1A is a simplified schematic view of a system for performing optical PCR according to some embodiments of the present invention.

[0009] Figure 1B is a flowchart illustrating a method for performing optical PCR according to an embodiment of the present invention.

[0010] Figures 2A through 2N is a simplified schematic cross-sectional view illustrating a fluid conduit extraction system according to an embodiment of the present invention.

[0011] Figures 3A through 3E is a simplified schematic cross-sectional view illustrating a fluid conduit extraction system according to an embodiment of the present invention.

[0012] Figure 4A and Figure 4B is a perspective view of a fluid conduit according to an embodiment of the present invention.

[0013] Figure 5 is a schematic view of a system for extracting nucleic acids according to an embodiment of the present invention.

[0014] Figures 6A through 6H is a simplified schematic cross-sectional view illustrating a fluid conduit extraction system according to an embodiment of the present invention. Detailed Description

[0015] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification, and in which are shown, by way of illustration, specific embodiments in accordance with the described embodiments. Although the embodiments are described in sufficient detail to enable those skilled in the art to practice the described embodiments, it is understood that these examples are not limiting; other embodiments may be used and changes may be made without departing from the spirit and scope of the described embodiments.

[0016] Conventional methods for separating nucleic acids for performing PCR generally require an initial nucleic acid separation step to separate the nucleic acids from materials that may interfere with the detection, hybridization, and / or amplification techniques used. A series of methods for separating nucleic acids are known, but these methods rely on a series of complex extraction and washing steps and are time-consuming and laborious to perform. For example, methods for separating nucleic acids from complex starting materials involve lysis of biological materials (possibly in the presence of protein-degrading enzymes), followed by several extractions with organic solvents, ethanol precipitation, centrifugation, and dialysis of the nucleic acids. Such methods are not only cumbersome and time-consuming to perform, but the relatively large number of steps required increases the risk of degradation, sample loss, or cross-contamination of samples when several samples are processed simultaneously.

[0017] Solid-phase extraction is a method for separating and extracting nucleic acids from biological samples. During solid-phase extraction, nucleic acids in the biological sample bind to a solid-phase substrate. In some embodiments, the solid-phase substrate can be a plurality of beads coated with silica or other materials. The solid-phase substrate (e.g., the beads) is then washed with an alcohol-based solution or other solution to remove the supernatant and loosely bound impurities from the nucleic acids bound to the solid-phase substrate. For example, prior to assaying the extracted nucleic acids, the solid-phase substrate is washed with a low-pH wash buffer or an alcohol-based wash buffer to remove any interfering substances. As used herein, a "low-pH wash buffer" can be a wash buffer having a pH less than 7 (e.g., less than 6, less than 5, less than 4, less than 3, or less than 2). To remove residual wash buffer components that can interfere with downstream processes, the surface of the solid-phase substrate is dried (e.g., using air) after one or more wash steps. After washing and drying the solid-phase substrate, the nucleic acids are eluted from the solid-phase substrate with a low ionic strength aqueous buffer.

[0018] Since many of the reagents used in the solid-phase extraction process interfere with downstream assays, it is typically necessary to remove any interfering substances prior to assaying the extracted nucleic acids. For example, guanidinium salts used in lysis buffers are typically removed so as not to interfere with downstream assays. Conventionally, several alcohol washes are performed to remove residual salts from the system. Additionally, the alcohol-based wash buffers (e.g., ethanol, propanol, etc.) frequently used in the wash steps are also removed in order to have a high extraction yield and to prevent interference with downstream assays. To remove the alcohol-based wash buffer, the process typically requires drying the solid-phase substrate, for example, by passing air over the solid-phase substrate, heating the solid-phase substrate, or applying a negative pressure to the solid-phase substrate. This introduces limitations to the procedure and is often difficult to implement in a cartridge, especially for cartridges that automate sample preparation. For example, sometimes high temperatures are used to evaporate ethanol; however, high temperatures can degrade nucleic acids, especially RNA. When air drying the solid-phase substrate, it takes a significant amount of time to remove or reduce the alcohol-based wash buffer to an acceptable level (typically less than 1 wt% in the eluate).

[0019] Embodiments of the present invention provide methods and systems for performing optical PCR. These methods and systems include eluting nucleic acids from a solid-phase substrate, which utilizes immiscible fluids to remove residual reagents or impurities (e.g., alcohol-based wash buffer) from the solid-phase substrate. In some embodiments, the method provides means for eluting nucleic acids bound to the surface of a solid-phase substrate as part of a nucleic acid purification process that uses immiscible fluids instead of air drying to remove residual reagents. In certain embodiments, an immiscible fluid (e.g., mineral oil) is utilized that does not extract nucleic acids from the solid-phase substrate but preferentially removes wash buffers (e.g., alcohols), salts, or other interfering substances. The use of such immiscible fluids accelerates the overall extraction process, results in a higher yield, and minimizes interference with downstream assays.

[0020] In addition, specific advantages have been found when the immiscible fluids mentioned above are used with specifically designed fluid conduits for the separation and extraction of nucleic acids. Conventional fluid conduits utilize conduits with a uniform cross-sectional area, where a solid-phase substrate can be captured in a certain region of the fluid conduit. In the fluid conduit, a series of lysis, wash, and elution buffers can pass through the conduit for the separation and extraction of nucleic acids. However, these methods do not provide sufficient contact between the lysis, wash, and elution buffers and the solid-phase substrate. Therefore, multiple washes are required to achieve the desired separation and extraction of nucleic acids. This results in a longer overall extraction process, a lower yield of nucleic acids, increased damage to the nucleic acids, and more interference with downstream assays. Due to the poor extraction and lower yield of nucleic acids during sample processing, amplification and detection during PCR can be affected.

[0021] In some embodiments, the present disclosure describes a system for performing optical PCR that has an improved process for the extraction and separation of nucleic acids. In some embodiments, the system includes a cartridge and a PCR device in communication with the cartridge. The cartridge can include a fluid conduit that includes a chamber for capturing a solid-phase substrate at a capture site within the fluid conduit. In some embodiments, the chamber has a larger cross-sectional area than the fluid conduit for receiving and holding the solid-phase substrate. For example, the solid-phase substrate can be a plurality of paramagnetic beads that include a silica coating for adhering a biological sample to the solid-phase substrate. The paramagnetic beads can be captured in the chamber, and a magnetic field can be applied to the chamber to hold the paramagnetic beads in the chamber. The magnetic field or magnetic field gradient can vary in intensity or orientation while maintaining sufficient force to keep the paramagnetic beads captured while allowing for better mixing with the sample, wash buffer, immiscible fluid, or elution buffer. The wash buffer can pass through the fluid conduit. When the wash buffer flows through the chamber containing the paramagnetic beads, the magnetic field can be directed in multiple directions (e.g., along the X-axis, Y-axis, or Z-axis) relative to the chamber to facilitate mixing of the wash buffer with the paramagnetic beads. In this way, there is increased contact between the wash buffer and the solid-phase substrate, thereby removing more residual components (e.g., supernatant) from the solid-phase substrate than would be achieved without such increased contact. The immiscible fluid can also pass through the fluid conduit. The magnetic field around the chamber can be selectively controlled (e.g., by changing its position, intensity, orientation, or gradient) to improve the contact between the working fluid and the solid-phase substrate to preferentially remove alcohols, salts, or other interfering substances. In some embodiments, other means of capturing the solid-phase substrate are envisioned, such as capturing beads by a weir in the conduit.

[0022] In some embodiments, a method for performing optical PCR includes providing a system. The system includes a cartridge for processing a biological sample and a PCR device in communication with the cartridge. The biological sample can be provided to a sample chamber of the cartridge. The method includes mixing the biological sample with a lysis buffer to produce a first fluid. In some aspects, the lysis buffer includes guanidine. In some embodiments, the method includes mixing the first fluid with a binding buffer (e.g., an alcohol-based binding buffer optionally including a solid-phase substrate) to produce a second fluid. The second fluid is contacted with a solid-phase substrate (e.g., paramagnetic beads) to bind nucleic acids to the solid-phase substrate. The solid-phase substrate is then washed with a fluid such as a low-pH wash buffer or an alcohol-based wash buffer to remove any interfering substances (e.g., salts, alcohols, etc.). The method includes introducing an immiscible fluid to remove the wash buffer and any other lysis substances from the solid-phase substrate prior to elution. The method includes transferring the eluted nucleic acids to the optical PCR device and performing PCR on the eluted nucleic acids. The optical PCR device can include an optical PCR cavity that includes a light-absorbing layer configured for photothermal conversion. For example, the eluted nucleic acids can be transferred via a channel to the optical PCR cavity of the optical PCR device. A light source can irradiate the light-absorbing layer of the optical PCR cavity for photothermal conversion to perform thermal cycling of the eluted nucleic acids.

[0023] These and other embodiments are discussed below with reference to the accompanying drawings. However, those skilled in the art will readily appreciate that the detailed description given herein with respect to these drawings is for explanatory purposes only and should not be construed as limiting.

[0024] Figure 1A FIG. illustrates a system for performing optical PCR. System 100 includes a cartridge 105 for processing a sample. In some embodiments, the sample is a biological sample (e.g., tissue, blood, serum, plasma, or spinal fluid). For example, cartridge 105 can be configured to extract and isolate nucleic acids from a biological sample for detection, hybridization, and / or amplification techniques. System 100 includes a PCR device 180 in communication with cartridge 105. In some embodiments, PCR device 180 can be coupled to cartridge 105. PCR device 180 is configured to perform optical PCR amplification on the processed sample received from cartridge 105.

[0025] The cartridge 105 may include a sample chamber 110. The sample chamber 110 is configured to receive a sample fluid. The sample chamber 110 may include an inlet 112 for receiving the sample. For example, the sample fluid may be provided to the sample chamber 110 via the inlet 112. The inlet 112 may be an injection port for receiving the sample fluid from a collection device (e.g., a hypodermic needle). In some embodiments, the sample fluid may be pre-treated before being provided to the sample chamber 110. For example, a whole blood sample may be centrifuged to separate blood cells and other solid components from serum or plasma. The separated serum or plasma may optionally be diluted or mixed with a reagent and provided to the sample chamber 110. In some embodiments, the cartridge 105 may include a pump 108 to supply the sample fluid (or other fluid) to areas of the cartridge 105. In some embodiments, the pump is configured for bubble mixing in the sample chamber.

[0026] The cartridge 105 may include a plurality of holes. The plurality of holes may be provided on the body of the cartridge 105. Each of the plurality of holes may include a recessed seat. The recessed seat may extend into the body of the cartridge 105. The recessed seats of the plurality of holes are configured to receive a storage container therein. For example, the storage container may be a blister pack that can be received in the hole. The blister pack may be configured to be pierced to release the fluid therein. In some embodiments, one or more of the plurality of holes may include a groove (e.g., on the outer circumference of the hole). The storage container may include an engagement member (e.g., a pointed tip) to engage with the groove to fix the storage container in the hole. For example, the storage container may snap-fit into the hole.

[0027] The cartridge 105 includes one or more of the plurality of holes that are in fluid communication with the sample chamber 110. For example, the cartridge 105 includes a first hole 120 and a second hole 125 that are in fluid communication with the sample chamber 110 via a channel. In some embodiments, the first hole 120 is configured to receive a storage container including a lysis buffer, and the second hole 125 is configured to receive a storage container including a solid phase substrate (e.g., paramagnetic beads). In some embodiments, the lysis buffer includes guanidine. The lysis buffer from the first hole 120 may be released from the storage container to provide the lysis buffer to the sample chamber 110 via the channel 121 to extract nucleic acids from the sample fluid. The solid phase substrate from the second hole 125 may be released from the storage container to provide the solid phase substrate to the sample chamber 110 via the channel 126 to bind the extracted nucleic acids to the solid phase substrate. The channels 121 and 126 may include valves or actuators to control the flow of fluid to the sample chamber 110. In some embodiments, the lysis of the biological sample and the binding of the extracted nucleic acids to the solid phase substrate may be completed in a pre-treatment step and then supplied to the sample chamber 110.

[0028] The cartridge 105 may include a fluid conduit 130 that includes a capture site 135. The fluid conduit 130 includes a capture site 135 for capturing a solid-phase substrate in the sample fluid within the fluid conduit 130. In some embodiments, the capture site 135 has a larger cross-sectional area than the fluid conduit 130 for receiving and retaining the solid-phase substrate in the sample fluid. For example, the capture site 135 may have an enlarged height, width, and / or length compared to the remainder of the fluid conduit 130. As discussed herein, a magnetic field source 136 may be located in a region adjacent to or within the capture site 135. For example, the magnetic field source 136 may be located in a region of the capture site 135 for capturing or immobilizing the solid-phase substrate. In some embodiments, a controller is coupled to the magnetic field source and configured to vary the strength, gradient, and / or orientation of the magnetic field source. For example, the controller may be configured to vary the position, strength, or magnetic field (e.g., width, height, or length of the magnetic field) of at least one of one or more magnetic field sources. In use, the sample fluid flows through the fluid conduit 130 past the magnetic field source 136 applied to the capture site 135, whereby the solid-phase substrate in the sample fluid is captured within the magnetic field while the other components of the sample fluid continue to flow along the fluid conduit 130, thereby removing all unbound molecules (e.g., supernatant) from the sample fluid. In some embodiments, the capture site 135 includes a shallow region (e.g., a weir). In some embodiments, the solid-phase substrate in the sample fluid is captured in the shallow region of the capture site 135.

[0029] The cartridge 105 includes one or more of a plurality of holes in fluid communication with the fluid conduit 130. For example, the cartridge 105 may include a third hole 140, a fourth hole 145, a fifth hole 150, and a sixth hole 155 that are in fluid communication with the fluid conduit 130 via a plurality of channels. In some embodiments, the third hole 140 is configured to receive a storage container including a first wash buffer, the fourth hole 145 is configured to receive a storage container including an immiscible fluid, the fifth hole 150 is configured to receive a storage container including a second wash buffer, and the sixth hole 155 is configured to receive a storage container including an elution buffer.

[0030] In some embodiments, system 100 may include one or more heaters (not shown) adjacent to capture site 135 of fluid conduit 130. For example, multiple heaters may be disposed below capture site 135 of fluid conduit 130. The one or more heaters may be configured to heat the components at capture site 135. For example, the one or more heaters may heat the sample fluid passing through capture site 135 as the sample fluid flows through fluid conduit 130, or the one or more heaters may heat the solid-phase substrate captured at capture site 135. In some embodiments, capture site 135 may be heated using one or more heaters while the sample fluid flows from the sample chamber to the fluid conduit, while the wash buffer flows through the fluid conduit, while the immiscible fluid flows through the fluid conduit, and / or while the elution buffer flows through the fluid conduit.

[0031] Cartridge 105 may include a first valve 160. The first valve 160 is configured to control the flow of fluid to fluid conduit 130. In some embodiments, the first valve 160 is configured to control the fluid to the first end of fluid conduit 130. The first valve 160 may be opened to allow the fluid sample to flow from sample chamber 110 to fluid conduit 130. In some embodiments, pump 108 may pressurize the flow of the sample fluid through the first valve 160 to fluid conduit 130. For example, the first valve 160 may be opened to allow the sample fluid to flow through fluid conduit 130 to capture the solid-phase substrate at capture site 135. After the solid-phase substrate is captured at capture site 135 of fluid conduit 130, the first valve 160 may be actuated to allow the first wash buffer from the third orifice 140 to flow to fluid conduit 130. The first wash buffer may remove all unbound molecules (e.g., supernatant) from the sample fluid. The first wash buffer including the unbound molecules may be supplied to waste chamber 175. In some embodiments, the first valve 160 may be actuated to sequentially allow the immiscible fluid from the fourth orifice 145 and the second wash buffer from the fifth orifice 150 to flow.

[0032] Cartridge 105 includes a second valve 165. In some embodiments, the second valve 165 is configured to control the flow of the elution buffer from the sixth orifice 155 to fluid conduit 130. In some embodiments, the elution buffer from the sixth orifice 155 may flow in a direction opposite to the other fluids supplied to fluid conduit 130. The second valve 165 may also be configured to supply the eluted nucleic acid to PCR device 180.

[0033] The PCR device 180 may include an optical PCR cavity 182 configured to receive the eluted nucleic acid from the fluid conduit 130. In some embodiments, the PCR devices described in U.S. Patent No. 11406983 to Son et al., U.S. Patent Publication No. 2021 / 0008562 to Son et al., and U.S. Patent Publication No. 2018 / 0080064 to Son et al. may be used in the system 100, and these patents are hereby incorporated by reference. The optical PCR cavity 182 may include a light absorption layer 184. The light absorption layer 184 may be configured to raise the temperature of the light absorption layer 184 according to the absorbed light so as to heat the eluted nucleic acid in the optical PCR cavity 182. For example, the light absorption layer includes a material having a light absorption rate greater than 50% (e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%). The light absorption layer 184 may include one or more metal layers. Non-limiting examples of metals that may be used are gold (Au), silver (Ag), nickel (Ni), titanium (Ti), chromium (Cr), germanium (Ge), palladium (Pd), ruthenium (Ru), tungsten (W), iridium (Ir), or platinum (Pt). In some cases, the light absorption layer is any alloy among the aforementioned alloys. The light absorption material may be a carbon-based material, non-limiting examples of which include carbon nanotubes, graphite, graphene, and / or graphene oxide. The light absorption layer may be composed of a coating such as an acrylic coating. The light absorption layer may include a mixture of a metal, a metal alloy, a carbon-based material, or a coating. In some cases, more than one layer of light absorption material may be used. One light absorption layer may absorb light from a light source and transmit the light not absorbed at the first light absorption layer to the second light absorption layer to generate heat. In some embodiments, the light absorption material used may be thin to maintain high heating and cooling rates. In some embodiments, the system 100 may include one or more light sources (not shown). The light source may be the main heat source of the optical PCR cavity. The light source may be a light-emitting diode (LED), a laser diode (LD), a tungsten filament lamp, a fluorescent lamp, a halogen lamp, a mercury lamp, a xenon lamp, a metal halide lamp, or a combination thereof.

[0034] In some embodiments, the optical PCR cavity may be surrounded by an external housing. The external housing may include one or more optical members, one or more heaters, and one or more temperature sensors. In some embodiments, one or more heaters are configured to heat the sample (e.g., the eluted nucleic acid) during optical PCR. In some embodiments, one or more heaters are configured to heat the sample before or after optical PCR.

[0035] Figure 1Bis a flow chart illustrating a method of performing optical PCR according to an embodiment of the present invention. Method 1000 includes providing a system (1010) for performing optical PCR. The system may include a cartridge configured to process a biological sample. The cartridge may include a sample chamber that includes an inlet configured to receive the biological sample. The cartridge may include a fluid conduit in communication with the sample chamber. The fluid conduit may include capture sites disposed along the length of the fluid conduit. In some embodiments, the capture sites may include one or more magnetic field sources disposed along the capture sites. The cartridge may include a plurality of wells. Each of the wells is configured to receive one of a plurality of fluids (e.g., lysis buffer, wash buffer, or elution buffer). A portion of the plurality of wells may be in fluid communication with the fluid conduit. The system may include a PCR device in communication with the cartridge. The PCR device includes an optical PCR cavity that includes a light absorption layer for performing PCR. In some embodiments, the system may be a Figure 1A system.

[0036] Method 1000 includes providing the biological sample to the inlet of the sample chamber (1020). For example, the biological sample may be injected into the inlet of the sample chamber. In some embodiments, the biological sample may be processed before being supplied to the sample chamber. For example, plasma or serum may be separated from whole blood and provided to the sample chamber.

[0037] Method 1000 includes flowing a lysis buffer from a first well of the plurality of wells to the sample chamber to extract nucleic acids from the biological sample (1030). The lysis buffer may be mixed with the biological sample to lyse biological structures in the biological sample, such as cells, viruses, organelles, or other nucleic acid-containing structures, to release nucleic acids and supernatant from the structures. In some aspects, the lysis buffer includes guanidine. The lysis buffer may lyse cells / organelles in the biological sample, and their contents (including nucleic acids) are released into the solution. In some embodiments, the biological sample is lysed using any method (e.g., by heating, with ultrasound, or with a suitable lysis fluid). In some embodiments, nucleic acids from the lysed biological sample may bind to a solid-phase substrate. The binding may occur in the sample fluid before the sample fluid is flowed, or at other points during the process or in another liquid medium.

[0038] Method 1000 includes flowing a solid-phase substrate from a second well of a plurality of wells to a sample chamber to bind extracted nucleic acids to the solid-phase substrate (1040). The solid-phase substrate may include a plurality of beads. In some embodiments, the beads may be magnetic beads or paramagnetic beads. In some embodiments, the beads may be composed of ceramics, polymers, or silica having a magnetic core or paramagnetic core. In some embodiments, the beads may be functionalized (e.g., with a silica or silica-like coating, with capture oligonucleotides, or with a charged polymer) to facilitate binding of nucleic acids, specific types of nucleic acids (e.g., DNA, RNA, or different nucleic acid size ranges), or specific nucleic acid sequences. In some embodiments, the solid-phase substrate may be directly supplied (e.g., injected) into the sample chamber.

[0039] The solid-phase substrate may be mixed with a lysed biological sample to produce a sample fluid. In some embodiments, the sample fluid includes nucleic acids bound to the solid-phase substrate. In this example, the sample fluid includes nucleic acids from the biological sample bound to the surface of the beads. In some embodiments, other components from the lysed cells in the biological sample may bind to the beads. As is known to those of ordinary skill in the art, there are many ways to bind nucleic acids in the sample fluid to the solid-phase substrate. Nucleic acids released from cells may bind to the solid-phase substrate, while the supernatant does not bind to the solid-phase substrate. The supernatant may include cell wall fragments, proteins, and other components within the cell. For example, the supernatant may be components within the biological sample that are not nucleic acids.

[0040] In some embodiments, bubble mixing may be performed by using a pump to mix the solid-phase substrate with the extracted nucleic acids from the biological sample. The solid-phase substrate and the extracted nucleic acids may be bubble mixed in the sample chamber. In some embodiments, the solid-phase substrate and the extracted nucleic acids may be mixed by advection, turbulent mixing, vortex methods, bubble mixing, microacoustics, stirring, or other mixing methods. The mixing may be achieved by an active mixing device or a passive mixing device.

[0041] Method 1000 includes flowing a sample fluid including the solid-phase substrate from the sample chamber to a fluid conduit (1050). For example, a valve may control the flow of the sample fluid from the sample chamber to the fluid conduit. In some embodiments, a pump is configured to move the sample fluid from the sample chamber to the fluid conduit. In some embodiments, the pump may force the sample fluid from the sample chamber to the fluid conduit. In some embodiments, the sample fluid is provided to a capture site through the fluid conduit. For example, the method includes flowing a sample fluid including nucleic acids bound to the solid-phase substrate through the fluid conduit to the capture site. The capture site may include a chamber within the fluid conduit. In the embodiment shown in FIG. 4, the chamber within the fluid conduit may have a larger cross-sectional area than the fluid conduit.

[0042] Method 1000 includes applying a magnetic field to capture a solid-phase substrate in a chamber (1060) of a fluid conduit. For example, when the capture site includes a chamber, a solid-phase substrate in a sample fluid can be captured in the chamber by applying a magnetic field (e.g., a local magnetic field) to the chamber volume. In particular, the sample fluid flows through the fluid conduit past the magnetic field applied to the chamber, whereby the solid-phase substrate is captured within the magnetic field while other components of the sample fluid continue to flow along the fluid conduit, thereby removing all unbound molecules (e.g., supernatant) from the sample fluid. In some embodiments, the chamber includes a shallow region (e.g., a weir). In some embodiments, the solid-phase substrate in the sample fluid is captured in the shallow region of the chamber.

[0043] Method 1000 includes flowing a wash buffer through the fluid conduit to remove impurities from the solid-phase substrate (1070). In some embodiments, one or more wash buffers pass through the fluid conduit. The wash buffer flows through the fluid conduit past the local magnetic field where the solid-phase substrate is captured within the magnetic field. The wash buffer extracts loosely bound molecules and residual reagents from the surface of the solid-phase substrate. In some embodiments, the wash buffer includes an alcohol. In some embodiments, the wash buffer includes methanol, ethanol, propanol, butanol, or a combination thereof. The wash buffer removes contaminants (e.g., supernatant, unbound cells, etc.) or residual reagents from or near the solid-phase substrate. This washing step can be repeated one or more times to remove all contaminants present on the surface of the solid-phase substrate.

[0044] Method 1000 includes flowing an immiscible fluid through the fluid conduit to remove residual sample fluid and / or wash buffer (1080). In some embodiments, the immiscible fluid is a water-immiscible fluid such as mineral oil. The immiscible fluid can remove any residual wash buffer from the surface of the solid-phase substrate. For example, after the wash buffer has passed through the fluid conduit, the immiscible fluid flows through the fluid conduit. The immiscible fluid flows within the fluid conduit past the local magnetic field where the solid-phase substrate, implemented as paramagnetic beads for example, is captured within the magnetic field. The flow of the immiscible fluid removes the wash buffer (e.g., ethanol or propanol) from the solid-phase substrate. In some embodiments, the immiscible fluid flows through the fluid conduit and removes the alcohol, salts (e.g., from a lysis buffer), or other interfering substances left after the wash buffer has passed, as described with respect to step 1030. Thus, the immiscible fluid removes any remaining contaminants from the solid-phase substrate. This step can be repeated one or more times to remove all wash buffer present on the surface of the solid-phase substrate.

[0045] Method 1000 includes flowing an elution buffer through a fluid conduit to elute nucleic acids (1090) from a solid-phase substrate. The elution buffer flows within the fluid conduit and through a local magnetic field where the solid-phase substrate is captured within the magnetic field. The elution buffer elutes (e.g., releases) nucleic acids from the surface of the solid-phase substrate. In some embodiments, the elution buffer may include water. In some embodiments, the elution buffer includes a low pH buffer. The eluted nucleic acids are picked up by the elution buffer and continue to flow along the fluid conduit for further biological processing and analysis. In some embodiments, the following fluids are passed through the fluid conduit: sample fluid, wash buffer, immiscible fluid, and elution buffer. In some embodiments, the following fluids or a subset of the following fluids are passed through the fluid conduit: sample fluid, air gap, wash buffer, air gap, immiscible fluid, air gap, and elution buffer. In some embodiments, the following fluids or a subset of the following fluids are passed through the fluid conduit: sample fluid, air gap, wash buffer, air gap, immiscible fluid, air gap, and elution buffer. In some embodiments, the following fluid mixture that may include solids is passed through the fluid conduit: solid-phase substrate, biological sample, lysis buffer, wash buffer, immiscible fluid, and elution buffer.

[0046] In some embodiments, the immiscible fluid passes through the fluid conduit before the elution buffer to prevent trace elements of the wash buffer (e.g., alcohol) from mixing with the elution buffer along the fluid conduit and / or the solid-phase substrate. The immiscible fluid may remove (e.g., completely remove) residual wash fluid from the fluid conduit and / or the solid-phase substrate, including residual contaminants remaining in the fluid conduit, such that drying of the fluid conduit or the solid-phase substrate is unnecessary. Thus, it is possible to directly elute nucleic acids after flowing the immiscible fluid through the fluid conduit.

[0047] Thus, in some embodiments, the method does not utilize a drying step after the passage of the wash fluid. This drying step utilized in conventional methods is time-consuming and cumbersome because additional processing steps (e.g., heating, vacuum, centrifugation) are utilized.

[0048] In some embodiments, the method includes heating one or more components within the fluid conduit. For example, the fluid conduit site may include one or more heaters adjacent to the capture site. The heaters may be disposed below the capture site of the fluid conduit 130. In some embodiments, the method includes heating the sample fluid passing through the capture site while the sample fluid flows through the fluid conduit. In some embodiments, the method includes heating the capture site while flowing the sample fluid from the sample chamber to the fluid conduit, flowing the wash buffer through the fluid conduit, flowing the immiscible fluid through the fluid conduit, and / or flowing the elution buffer through the fluid conduit.

[0049] Method 1000 includes transferring the eluted nucleic acid to a PCR device (1095). The PCR device is configured to perform nucleic acid amplification by polymerase chain reaction. In some embodiments, the PCR device includes an optical PCR chamber. The optical PCR chamber includes a light absorption layer disposed on a substrate. In some embodiments, the substrate is a transparent substrate. For example, the transparent substrate may allow 50% or more (e.g., 50% or more, 60% or more, 70% or more, 75% or more, or 80% or more) of the light to pass through the substrate to reach the light absorption layer. The light absorption layer may be configured to raise the temperature of the light absorption layer according to the absorbed light, so as to heat the fluid sample in the optical PCR chamber for nucleic acid amplification. In some embodiments, the light absorption layer has a light absorption rate of 50% or more (e.g., 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more) of light of a certain wavelength (e.g., visible light, UV, infrared, etc.).

[0050] Method 1000 includes performing PCR (1096) on the eluted nucleic acid in the optical PCR chamber. In some embodiments, the system may include a light source for guiding light towards the light absorption layer of the optical PCR chamber. For example, the light source may be one or more light emitting diodes. Alternative light sources may include: laser diodes (LDs), tungsten filament lamps, fluorescent lamps, halogen lamps, mercury lamps, xenon lamps, metal halide lamps, or any combination of the foregoing light sources. The light source may guide light towards the optical PCR chamber for nucleic acid amplification by PCR. In some embodiments, the light is irradiated onto the light absorption layer through the substrate. The light absorption layer is configured to perform photothermal conversion for PCR thermal cycling. The absorbed light contributes to the photothermal heating of the light absorption layer for the thermal cycling of PCR.

[0051] It should be appreciated that Figure 1B the specific steps illustrated in provide a particular method of extracting nucleic acid according to an embodiment of the present invention. According to alternative embodiments, other sequences of steps may also be performed. For example, alternative embodiments of the present invention may perform the steps outlined above in a different order. Additionally, Figure 1B the individual steps illustrated in may include multiple sub-steps, which may be performed in various sequences suitable for the individual steps. Additionally, additional steps may be added or removed depending on the specific application. Those of ordinary skill in the art will recognize many variations, modifications, and alternatives.

[0052] Figures 2A through 2NFIG. 0 is a simplified schematic cross-sectional view of a fluid conduit extraction system according to an embodiment of the present invention. In some embodiments, the system 200 for extracting nucleic acids includes a fluid conduit 205. For example, the fluid conduit 205 may be a microfluidic channel. The fluid conduit 205 may include a delivery device for supplying the fluids described herein through the fluid conduit. For example, the delivery device may be a pump (not shown) for applying pressure to the fluid conduit 205 to move fluid through the fluid conduit 205. The pump may be attached to the fluid conduit for providing various fluids through the fluid conduit. In some embodiments, the fluid passing through the fluid conduit 205 passes in the form of slugs. For example, a slug may be a volume of fluid that fills the entire cross-section of the fluid conduit. In some embodiments, the fluid conduit 205 may include a tube having an airtight seal such that a vacuum exists in the tube. The pump may apply pressure to move the slugs of fluid through the fluid conduit 205.

[0053] Figure 2A FIG. 4 shows a slug of sample fluid 215 flowing within the fluid conduit 205. In some embodiments, the slugs of sample fluid 215 are aspirated through the fluid conduit 205 at various time points and / or at various speeds. The sample fluid 215 includes target molecules (e.g., nucleic acids) from a biological sample (e.g., whole blood) that are adhered to a solid-phase substrate 216 (e.g., paramagnetic beads). In some embodiments, the biological sample may be mixed with the solid-phase substrate before being supplied to the fluid conduit 205. In some embodiments, the biological sample may be mixed with one or more buffer solutions and the solid-phase substrate to form the sample fluid 215. For example, the buffer solution may include a lysis buffer and / or a binding buffer. The lysis buffer may lyse the cells / organelles in the biological sample, and their contents (including nucleic acids) are released into the solution. The binding buffer may facilitate the binding of nucleic acids to the solid-phase substrate 216. In some embodiments, the biological sample is lysed using any method (e.g., by heating, with ultrasound, or with a suitable lysis fluid). The target molecules from the lysed biological sample may bind to the solid-phase substrate 216. The binding may occur in the sample fluid before the sample fluid is made to flow, or at other points during the process or in another liquid medium.

[0054] In some embodiments, the solid-phase substrate 216 can be any suitable substrate for attracting and adhering target molecules from a biological sample. For example, the solid-phase substrate 216 can be one or more of paramagnetic beads, magnetic beads, glass beads, glass fibers, glass membranes, etc. In some embodiments, the solid-phase substrate 216 comprises a plurality of paramagnetic beads optionally comprising a coating (e.g., silica or capture oligonucleotide). The solid-phase substrate 216 can be used in a system similar to the systems outlined herein to assist in the assembly of nucleic acid structures. The solid-phase substrate 216 provides a large surface-to-volume ratio useful in exposing relevant binding chemistries. In some embodiments, the solid-phase substrate 216 can be used to separate and enrich target cells from a biological sample.

[0055] Figure 2B Shows the solid-phase substrate 216 captured within the capture site 220 of the fluid conduit. In some embodiments, the magnetic field source 210 is adjacent to the exterior of the capture site 220 of the fluid conduit 205. For example, the magnetic field source 210 can be located in a predetermined region for capturing or immobilizing the solid-phase substrate 216 in the sample fluid. As Figure 4A and Figure 4B shown, the fluid conduit can include a chamber that forms the capture site. The chamber can be located at a predetermined position in the fluid conduit 205 and is configured to capture the solid-phase substrate by applying a magnetic field from the magnetic field source 210. In some embodiments, the fluid conduit 205 includes a chamber having a cross-sectional area that is larger than the average cross-sectional area of the fluid conduit. For example, Figure 4A and Figure 4B shows a chamber 410 that can have an enlarged height, width, and / or length compared to the rest of the fluid conduit. In some embodiments, the chamber can be cylindrical, hexagonal, rectangular parallelepiped, or spherical. The solid-phase substrate 216 can be immobilized within the chamber of the fluid conduit 205.

[0056] Figure 2C Shows the supernatant separated from the sample fluid 215. The target biomolecules bound to the solid-phase substrate 216 in the sample fluid 215 are captured in the fluid conduit 205 using the magnetic field source 210. The unbound portion of the sample fluid 215 continues through the fluid conduit 205. The unbound portion of the sample fluid 215, including the supernatant, is then discarded. For example, in embodiments using paramagnetic beads, a slug of the sample fluid 215 continues to flow within the fluid conduit 205 while the paramagnetic beads with the bound target biomolecules remain captured in the fluid conduit 205 by the magnetic field source 210.

[0057] Figure 2DShows a slug of wash buffer 225 introduced into fluid conduit 205. The wash buffer 225 flows through the fluid conduit 205. The wash buffer 225 flows over and through the captured solid-phase substrate 216 in the fluid conduit 205. In some embodiments, the wash buffer 225 includes an alcohol. For example, the wash buffer 225 may include ethanol, propanol, or other alcohols or combinations thereof. One or more slugs of wash buffer 225 may be introduced into the fluid conduit 205. In some embodiments, a slug of fluid may be followed by a slug of air (e.g., an air gap between slugs of fluid). As Figure 2E shown, the slug of wash buffer 225 flows within the fluid conduit 205 through a local magnetic field where the solid-phase substrate is captured within the magnetic field while other components of the slug of wash buffer 225 continue to flow along the fluid conduit. The slug of wash buffer 225 removes loosely bound molecules and residual reagents from the surface of the solid-phase substrate. In other words, the slug of wash buffer 225 clears contaminants from the solid-phase substrate.

[0058] Figure 2F Shows a slug of immiscible fluid 230 introduced into fluid conduit 205. The slug of immiscible fluid 230 flows through the fluid conduit 205 and passes over and / or through the solid-phase substrate 216. The slug of immiscible fluid 230 may include mineral oil, silicone oil, hexadecane, paraffin oil, fluorinated liquids, fluorinated oils, and mixtures thereof. As Figure 2G shown, the slug of immiscible fluid 230 passes through the capture site including the solid-phase substrate 216, which is captured by the magnetic field generated by the magnetic field source 210. The immiscible fluid may remove any residual wash buffer from the surface of the fluid conduit 205 and the solid-phase substrate 216.

[0059] Figure 2H and Figure 2I Shows a magnetic field source 212 positioned at a second location relative to the fluid conduit 205. In some embodiments, the magnetic field source 210 may be reoriented, as Figure 2H and Figure 2I shown. The magnetic field source 212 may generate a magnetic field that varies (e.g., oscillates) in multiple directions around the fluid conduit 205 to move the solid-phase substrate 216 relative to the fluid conduit 205. For example, magnets may be disposed along discrete regions of the fluid conduit 205, and a controller coupled to the magnetic field source is configured to change the intensity, gradient, and / or orientation of the magnetic field source. In some embodiments, the magnetic field source 212 that captures the solid-phase substrate 216 may be adjusted in one or more directions to facilitate mixing of one or more fluids (e.g., wash buffer or immiscible fluid) with the solid-phase substrate 216. As Figure 2HAs shown, the magnetic field source 212 can be translated along the Y axis (e.g., up and down) to move the solid-phase substrate 216 within the fluid conduit 205. As Figure 2I shown, the magnetic field 214 can be translated along the X axis (e.g., left and right) to move the solid-phase substrate 216 within the fluid conduit 205. The magnetic field 214 can be translated along the Z axis (e.g., forward and backward) to move the solid-phase substrate 216 within the fluid conduit 205. In some embodiments, the controller moves (e.g., oscillates) the magnetic field source relative to the chamber in a horizontal or vertical direction (e.g., XYZ directions). Figure 2J shows immiscible fluid 230 including any residual wash buffer or contaminants, which flows out of the fluid conduit. The magnetic field sources 210 and 212 (or other magnetic field sources) can be present simultaneously.

[0060] FIG. 2K shows a slug of elution buffer 240 introduced into the fluid conduit 205. The elution buffer 240 flows over and through the solid-phase substrate 216 ( Figure 2L ). The slug of elution buffer 240 releases the target biomolecules (e.g., nucleic acids) bound to the solid-phase substrate 216. The target biomolecules released from the solid-phase substrate 216 can be taken up or absorbed by the slug of elution buffer 240. Figure 2M shows a time-varying magnetic field source 210 located at a position along the fluid conduit 205 to facilitate contact between the elution buffer and the solid-phase substrate 216. Figure 2N shows elution buffer 240 including the eluted target biomolecules, which leaves the fluid conduit 205 for further processing and / or analysis.

[0061] Figures 3A through 3E is a simplified schematic cross-sectional view illustrating a fluid conduit extraction system according to an embodiment of the present invention. In some embodiments, the system for extracting nucleic acids includes a fluid conduit 305. For example, the fluid conduit 305 can be a microfluidic channel. The fluid conduit 305 includes a chamber 310 for holding a solid-phase substrate within a predetermined region of the fluid conduit. The chamber 310 can be located at a predetermined site within the fluid conduit 305.

[0062] Figure 3A shows a slug of sample fluid 320 when the sample fluid 320 begins to flow through the fluid conduit 305. In some embodiments, the slug of sample fluid 320 includes target biomolecules bound to the solid-phase substrate. The slug of sample fluid 320 traverses the fluid conduit 305 at a certain rate. For example, a pump (not shown) can apply variable pressure to regulate the speed of the fluid through the fluid conduit 305. Figure 3BShows a solid-phase substrate captured in a sample fluid 320 within a chamber 310, which chamber 310 may also be referred to as a capture site. In some embodiments, one or more magnets are disposed around a predetermined region of the chamber 310. The one or more magnets can be selectively controlled to change or move a magnetic field source 315 to different positions relative to the chamber 310. When a slug of the sample fluid 320 passes through the fluid conduit 305, the magnetic field source 315 can be applied to capture the solid-phase substrate in the slug of the sample fluid 320 at the capture site. In this way, the solid-phase substrate in the sample fluid 320 is captured in the chamber 310.

[0063] Figure 3C Shows a supernatant 325 separated from the sample fluid 320. A target biomolecule bound to the solid-phase substrate in the sample fluid 320 is captured in the chamber 310 of the fluid conduit 305 using a magnetic field source 315, which magnetic field source 315 can be supplemented by a physical structure as illustrated in FIG. 4. The supernatant 325 (e.g., the unbound portion of the sample fluid) continues through the fluid conduit 305. Figure 3D Shows a slug of immiscible fluid 330 introduced into the fluid conduit 305, and Figure 3E Shows a slug of immiscible fluid 330 exiting the fluid conduit 305. In some embodiments, after one or more wash buffers flow through the fluid conduit 305, a slug of the immiscible fluid 330 flows through the fluid conduit. In some embodiments, one or more slugs of the immiscible fluid 330 flow through the fluid conduit alternately with one or more slugs of the wash buffer, where the last slug is the immiscible fluid to remove any residual wash buffer. In some embodiments, the immiscible fluid includes mineral oil, silicone oil, hexadecane, paraffin oil, fluorinated fluid, or a mixture thereof. The slug of the immiscible fluid 330 flows through the fluid conduit 305 and passes over and through the solid-phase substrate captured in the chamber 310 of the fluid conduit 305 by the magnetic field. In some embodiments, after a wash buffer (e.g., an ethanol slug) has passed through the fluid conduit 305, a slug of the immiscible fluid 330 is introduced into the fluid conduit 305. The slug of the immiscible fluid 330 removes trace elements of the wash buffer from the surface of the fluid conduit and / or the solid-phase substrate. Subsequently, a slug of an elution buffer is introduced into the fluid conduit 305 to release the target biomolecule from the solid-phase substrate into the elution buffer.

[0064] After the wash step and before the elution step, flowing the immiscible fluid through the fluid conduit results in the unexpected advantage of reducing or eliminating the process of drying the fluid conduit and / or the solid-phase substrate to remove residual wash fluid, which process is typically necessary using conventional methods. By eliminating the need to dry the fluid conduit and / or the solid-phase substrate, there is the unexpected advantage of significantly shortening the duration of nucleic acid separation and extraction.

[0065] Figure 4A and Figure 4B is a perspective view of a fluid conduit according to an embodiment of the present invention. In some embodiments, a system for extracting nucleic acids includes a fluid conduit 405. For example, the fluid conduit 405 can be a microfluidic channel. The fluid conduit 405 includes a chamber 410. The chamber 410 can have a depth measured along the Y-axis direction of the fluid conduit 405 for holding a solid-phase substrate within a capture site of the fluid conduit 405. A magnetic field source 415 can be present in a region surrounding part or all of the chamber 410. In some embodiments, the magnetic field strength, orientation, gradient strength, and gradient orientation can vary over time while still maintaining sufficient force on the solid-phase substrate to keep the solid-phase substrate captured within the fluid conduit 405. In some embodiments, the magnetic field generated by the magnetic field source 415 can move in one or more predetermined directions around the circumference of the chamber 410 to facilitate mixing of a fluid (e.g., a wash buffer, an immiscible fluid, or an elution buffer) with the solid-phase substrate captured therein.

[0066] In some embodiments, the chamber 410 includes a larger cross-sectional area than the fluid conduit 405 to capture the solid-phase substrate therein. For example, the fluid conduit 305 includes a chamber 410 having a cross-sectional area larger than the average cross-sectional area of the fluid conduit 405. In one example, compared to the remainder of the fluid conduit 405, the chamber 410 of the fluid conduit 405 can have an enlarged height measured along the Y-axis of the fluid conduit 405, an enlarged width measured along the Z-axis of the fluid conduit 405, and / or an enlarged length measured along the X-axis of the fluid conduit 405. For example, Figure 4A shows that along the Y-axis of the fluid conduit 405, the height (h1) of the fluid conduit 405 is less than the height (h2) of the chamber 410. Similarly, when measured along the Z-axis of the fluid conduit, the width of the fluid conduit 405 can be less than the width of the chamber 410. In some embodiments, the shape of the chamber 410 can be cylindrical, hexagonal, cuboid, or spherical. The cross-sectional area of the chamber 410 can be at least 5% larger than the average cross-sectional area of the fluid conduit, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% larger.

[0067] In some embodiments, the magnetic field source 415 surrounds part or all of the chamber 410. For example, the magnetic field source 415 can be located at any position along the length or width of the chamber 410 for capturing or immobilizing a solid-phase substrate. In some embodiments, magnets can be disposed along discrete regions of the chamber 410. A controller can be coupled to one or more magnets. The controller can be configured to move or oscillate the magnetic field source 415 generated by the one or more magnets. The magnetic field source 415 can be moved in any direction to facilitate mixing of a fluid (e.g., wash buffer or immiscible fluid) with the solid-phase substrate. For example, the magnetic field source 415 can be translated along the Y axis (e.g., up and down) to move the solid-phase substrate within the chamber 410 of the fluid conduit 405. In another example, the magnetic field source 415 can be translated along the X axis (e.g., left and right) to move the solid-phase substrate within the chamber 410 of the fluid conduit 405. When a fluid (e.g., wash buffer or immiscible fluid) passes through the chamber 410, movement of the magnetic field source 415 to different regions of the chamber 410 facilitates mixing and contact with the solid-phase substrate.

[0068] Exemplary methods for separating and extracting nucleic acids are provided. When performing solid-phase extraction with magnetic silica beads, the exemplary method utilizes mineral oil as the final wash prior to elution.

[0069] A 250 μl sample containing 100 copies / ml of SARS-CoV2 RNA, carrier RNA, and total human RNA1 (1 ng / μl) in a viral transport medium is provided. The sample is mixed with 500 μl of 6M GU-SCN (lysis buffer) and 500 μl of 100% ethanol containing Cytiva Sera-Sil 700 nm paramagnetic beads. After a 1-minute incubation, the beads are magnetically separated from the supernatant in a microfluidic channel. The beads are then washed twice with 500 μl of a low ionic strength 80% ethanol wash buffer. The beads are then washed with 250 μl of PCR-grade mineral oil. The nucleic acids are eluted with 16 μl of TE buffer and the nucleic acids are assayed by rt-qPCR. The extraction is also performed in tubes. It was found that oil washing reduced the time required for nucleic acid extraction by more than 5 minutes compared to air-drying the beads after the final wash. Additionally, the eluate had a higher output RNA concentration: an average of 970 copies / ml for oil compared to an average of 360 copies / ml for air-drying.

[0070] Figure 5FIG. 0 is a schematic diagram of a system for extracting nucleic acids according to an embodiment of the present invention. System 500 is configured to extract nucleic acids from a biological sample. System 500 includes a fluid conduit 505. Fluid conduit 505 includes a chamber 510 for receiving one or more fluids. Chamber 510 is disposed at a location along the length of fluid conduit 505. The chamber may have a cross-sectional area greater than the average cross-sectional area of the fluid conduit. Fluid conduit 505 includes a magnetic field source 515 coupled to a controller 520. Controller 520 may be coupled to magnetic field source 515. In some embodiments, fluid conduit 505 includes a plurality of magnetic field sources along discrete regions of chamber 510, each of which is controlled by controller 520. Controller 520 is configured to move (e.g., oscillate) the magnetic field source (e.g., turn the magnetic field on or off). For example, controller 520 may be configured to change (e.g., oscillate) magnetic field source 515 in any direction within chamber 510 between two different magnetic field sources. In some embodiments, system 500 includes a pump 525 configured to pump a plurality of fluids through fluid conduit 505.

[0071] Figures 6A through 6H FIG. 4 is a simplified schematic cross-sectional view illustrating a fluid conduit extraction system according to another embodiment of the present invention. As Figure 6A shown, a system 600 for extracting nucleic acids includes a fluid conduit 605. Fluid conduit 605 includes a chamber 610 for holding a solid phase substrate within a predetermined region of the fluid conduit. One or more magnets 615 are disposed around or within chamber 610. In some embodiments, one or more magnets 615 apply a magnetic field to a region of the chamber that is less than at least one dimension of the chamber. For example, the magnetic field generated by one or more magnets 615 may be less than the length of chamber 610 to capture the solid phase substrate within chamber 610. In some embodiments, the length, height, and width of the magnetic field applied by one or more magnets 615 may be less than, greater than, or approximately the same size as chamber 610. Figure 6A FIG. 8 shows a slug of sample fluid 620 as it begins to flow from left to right through fluid conduit 605. In some embodiments, sample fluid 620 flows from right to left through fluid conduit 605. In some embodiments, the slug of sample fluid 620 includes target biomolecules bound to a solid phase substrate.

[0072] In some embodiments, the fluid conduit 605 may include a fluid capture region 650. The fluid capture region 650 may be adjacent to or within the chamber 610. For example, the fluid capture region 650 may be a region that tapers from the chamber 610 into the fluid conduit 605. In some embodiments, the fluid capture region 650 is a cavity within the chamber 610. The fluid capture region 650 is configured to capture or hold an immiscible fluid. As further discussed herein, the fluid capture region 650 may be used to capture any immiscible fluid such that it does not block the flow of the elution buffer over the solid phase substrate.

[0073] Figure 6B Shows a solid phase substrate captured in the sample fluid 620 within the chamber 610, which may also be referred to as a capture site. One or more magnets 615 may be selectively controlled to vary or move the magnetic field from the magnets 615 to different positions relative to the chamber 610. As a slug of the sample fluid 620 passes through the fluid conduit 605, the magnets 615 may apply a magnetic field to capture the solid phase substrate within the slug of the sample fluid 620 at the capture site. In this manner, the solid phase substrate within the sample fluid 620 is captured in the chamber 610.

[0074] Figure 6C Shows the supernatant 625 separated from the sample fluid 620. The target biomolecules that are bound to the solid phase substrate within the sample fluid 620 are captured in the chamber 610 of the fluid conduit 605 using one or more magnets 615, which may be supplemented by a physical structure as illustrated in FIG. 4. The supernatant 625 (e.g., the unbound portion of the sample fluid) continues through the fluid conduit 605. Figure 6D Shows a slug of immiscible fluid 630 introduced into the fluid conduit 605. In some embodiments, the immiscible fluid directly displaces the sample fluid or other wash buffer. In other embodiments, there is an air gap between the two fluids. In some embodiments, the slug of immiscible fluid 630 moves through the fluid conduit 605 from left to right or vice versa.

[0075] Figure 6EShows a slug of immiscible fluid 630 in the filling chamber 610. In some embodiments, after one or more wash buffers flow through the fluid conduit 605, a slug of immiscible fluid 630 flows through the fluid conduit. In some embodiments, one or more slugs of immiscible fluid 630 alternate with one or more slugs of wash buffer flowing through the fluid conduit, where the last slug is immiscible fluid to remove any residual wash buffer. The slug of immiscible fluid 630 flows through the fluid conduit 605 and passes over and through the solid-phase substrate trapped in the chamber 610 of the fluid conduit 605 by a magnetic field. In some embodiments, a slug of immiscible fluid 630 is introduced into the fluid conduit 605 after the wash buffer (e.g., ethanol slug) has passed through the fluid conduit 605. The slug of immiscible fluid 630 removes trace elements of the wash buffer from the surface of the fluid conduit and / or the solid-phase substrate.

[0076] Figure 6F Shows a slug of elution buffer 640 introduced into the fluid conduit 605 to displace the immiscible fluid and release the target biomolecule from the solid-phase substrate into the elution buffer. In this embodiment, the slug of elution buffer 640 can flow through the fluid conduit 605 from right to left. In some embodiments, the slug of elution buffer 640 is introduced in a direction opposite to the flow direction of the immiscible fluid 630. In other embodiments, the elution buffer and the immiscible fluid are introduced in the same direction. The elution buffer remains in contact with the solid-phase substrate in the chamber 610 to allow elution of the target molecule.

[0077] Figure 6G Shows a fluid capture region 650 adjacent to or within the chamber 610 that captures the immiscible fluid 630. The fluid capture region 650 can be used to capture any additional immiscible fluid 630 so that it does not block the flow of the elution buffer over the solid-phase substrate. The fluid capture region 650 can be disposed proximal to the captured solid-phase substrate with respect to the flow of the elution buffer. In some embodiments, the fluid capture region 650 includes a region of the chamber 610 or the fluid conduit 605 having a larger cross-sectional area than the remainder of the chamber 610 or the fluid conduit 605. In some embodiments, the fluid capture region 650 is located in the top region of the chamber 610 or the fluid conduit 605 such that gravity will act to force the lower density immiscible fluid into the fluid capture region 650. In some embodiments, the fluid capture region 650 is surface-treated to preferentially retain the immiscible fluid compared to the elution buffer (e.g., using a lipophilic coating within a hydrophilic chamber / conduit when using an oil-based immiscible fluid).

[0078] Figure 6HThe reintroduction of immiscible fluids (from left to right) is shown to push the elution buffer containing eluent 660 into the catheter for delivery to other parts of the system. In some embodiments, there is an air gap between the two fluids. In some embodiments, air is used to push the elution buffer out of the chamber and into the catheter.

[0079] For purposes of explanation, the foregoing description uses specific nomenclature to provide a thorough understanding of the described embodiments. However, it will be apparent to one of ordinary skill in the art that specific details are not required in order to practice the described embodiments. Accordingly, the foregoing description of specific embodiments is presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the described embodiments to the precise forms disclosed. Many modifications and variations will be apparent to one of ordinary skill in the art in light of the above teachings.

Claims

1. A system for performing PCR, the system comprising: a cartridge, comprising: a sample chamber, comprising an inlet configured to receive a biological sample; a fluid conduit in communication with the sample chamber, the fluid conduit comprising a capture site disposed along a length of the fluid conduit, the capture site having a cross-sectional area greater than an average cross-sectional area of the fluid conduit; a plurality of holes disposed in a body of the cartridge, wherein each of the holes is configured to receive one of a plurality of fluids, and wherein a portion of the plurality of holes is in fluid communication with the fluid conduit; one or more magnetic field sources disposed adjacent to the capture site; and a PCR device coupled to the cartridge, wherein the PCR device comprises an optical PCR cavity, the optical PCR cavity comprising a light absorption layer.

2. The system according to claim 1, wherein Each of the plurality of holes is configured to receive a storage container comprising one of the plurality of fluids, wherein the storage container is configured to be pierced to release the fluid therein.

3. The system according to claim 2, wherein, The plurality of fluids comprises one or more of a lysis buffer, a wash buffer, an alcohol, an immiscible fluid, or an elution buffer.

4. The system according to claim 1, wherein At least one of the plurality of holes comprises a solid phase substrate.

5. The system according to claim 1, wherein, The plurality of holes comprises: a first hole configured to receive a lysis buffer; a second hole configured to receive a first wash buffer; a third hole configured to receive a solid phase substrate; a fourth hole configured to receive an immiscible fluid; a fifth hole configured to receive a second wash buffer; and a sixth hole configured to receive an elution buffer.

6. The system according to claim 5, wherein The first hole and the third hole are in fluid communication with the sample chamber.

7. The system according to claim 5, wherein, The second hole, the fourth hole, the fifth hole, and the sixth hole are in fluid communication with the fluid conduit.

8. The system according to claim 1, wherein The cartridge further comprises a waste chamber.

9. The system according to claim 1, further comprising a plurality of valves, wherein, A first valve of the plurality of valves is configured to provide fluid from the sample chamber or the plurality of holes to the fluid conduit.

10. The system according to claim 9, wherein, A second valve is configured to provide fluid from the fluid conduit to the PCR device.

11. The system according to claim 1, further comprising a pump in communication with the sample chamber, and wherein, The pump is configured to provide pressure to move a biological sample through the fluid conduit.

12. The system according to claim 1, wherein, The capture site has a cross-sectional area that is at least 10% greater than the average cross-sectional area of the fluid conduit.

13. The system according to claim 12, wherein, At least one of the one or more magnetic field sources is configured to move in a direction around the capture site of the fluid conduit.

14. The system according to claim 1, wherein The shape of the capture site is cylindrical, hexagonal, cuboid, or spherical.

15. The system according to claim 1, further comprising a controller coupled to the one or more magnetic field sources, and wherein, The controller is configured to change a position, intensity, or field of at least one of the one or more magnetic field sources.

16. The system according to claim 1, wherein, The light absorption layer comprises a material having a light absorption rate greater than 50%.

17. The system according to claim 1, further comprising one or more heaters positioned adjacent to the capture site.

18. The system according to claim 17, wherein, The one or more heaters are configured to heat components in the capture site.

19. The system according to claim 1, wherein, The optical PCR cavity is surrounded by an outer housing.

20. The system according to claim 19, wherein, The outer housing comprises one or more optical components, one or more heaters, and one or more temperature sensors.

21. The system according to claim 20, wherein The one or more heaters are configured to heat a sample during optical PCR.

22. A method for performing PCR, the method comprising: A system for processing a biological sample is provided, the system comprising: a cartridge, comprising: a sample chamber comprising an inlet configured to receive a biological sample; a fluid conduit in communication with the sample chamber, the fluid conduit comprising a capture site disposed along a length of the fluid conduit, the capture site having a cross-sectional area greater than an average cross-sectional area of the fluid conduit; and a plurality of holes disposed in a body of the cartridge, wherein each of the holes is configured to receive one of a plurality of fluids, and wherein a portion of the plurality of holes is in fluid communication with the fluid conduit; one or more magnetic field sources disposed adjacent to the capture site; and a PCR device in communication with the cartridge, wherein the PCR device comprises an optical PCR cavity, the optical PCR cavity comprising a light absorption layer configured for photothermal conversion; providing a biological sample to the inlet of the sample chamber; flowing a lysis buffer from a first one of the plurality of holes to the sample chamber to extract nucleic acids from the biological sample; flowing a solid phase substrate from a second one of the plurality of holes to the sample chamber to produce a sample fluid comprising the extracted nucleic acids bound to the solid phase substrate; flowing the sample fluid from the sample chamber to the fluid conduit; applying a magnetic field to capture the solid phase substrate in the sample fluid at the capture site of the fluid conduit; flowing a wash buffer through the fluid conduit to remove impurities from the solid phase substrate; flowing an immiscible fluid through the fluid conduit to remove residual sample fluid and / or wash buffer; flowing an elution buffer through the fluid conduit to elute nucleic acids from the solid phase substrate; transferring the eluted nucleic acids to the optical PCR cavity; and performing PCR on the eluted nucleic acids in the optical PCR cavity.

23. The method according to claim 22, further comprising mixing the lysis buffer and the biological sample to extract nucleic acids from the biological sample.

24. The method according to claim 22, further comprising mixing the biological sample and the solid phase substrate to bind the nucleic acids to the solid phase substrate.

25. The method according to claim 23 or 24, wherein Mixing comprises performing bubble mixing using a pump.

26. The method according to claim 22, wherein, Flowing the immiscible fluid occurs after flowing the wash buffer.

27. The method according to claim 22, wherein The wash buffer comprises an alcohol-based wash buffer.

28. The method according to claim 27, wherein The alcohol-based wash buffer comprises ethanol or propanol.

29. The method according to claim 22, wherein, The method does not include flowing air through the fluid conduit to dry the fluid conduit or the solid phase substrate.

30. The method according to claim 22, further comprising applying a time-varying magnetic field to move the solid-phase substrate in the fluid conduit, wherein, The magnetic field is configured to maintain a sufficient force on the solid phase substrate to hold the solid phase substrate captured during at least one of: flowing the wash buffer through the fluid conduit; flowing the immiscible fluid through the fluid conduit; or flowing the elution buffer through the fluid conduit.

31. The method according to claim 22, wherein, The capture site has a cross-sectional area that is at least 10% greater than the average cross-sectional area of the fluid conduit.

32. The method according to claim 22, wherein The capture site is characterized by a hexagonal shape.

33. The method according to claim 22, wherein, The system further comprises one or more heaters positioned adjacent to the capture site.

34. The method according to claim 33, wherein, The method further includes heating the capture site using the one or more heaters during at least one of the following: flowing the sample fluid from the sample chamber to the fluid conduit; flowing the wash buffer through the fluid conduit; flowing the immiscible fluid through the fluid conduit; and / or flowing the elution buffer through the fluid conduit.

35. The method of claim 22, further comprising changing the magnetic field during at least one of the following to move the solid-phase substrate in the capture site: flowing the wash buffer through the fluid conduit; flowing the immiscible fluid through the fluid conduit; or flowing the elution buffer through the fluid conduit.

36. The method according to claim 22, wherein, Transferring the eluted nucleic acid from the solid-phase substrate to the elution buffer, wherein flowing the elution buffer includes flowing the eluted nucleic acid away from the capture site.

37. The method according to claim 22, wherein, The immiscible fluid includes one or more of mineral oil, silicone oil, hexadecane, paraffin oil, fluorinated liquid, fluorinated oil, or a mixture thereof.

38. The method according to claim 22, wherein, The solid-phase substrate includes a plurality of beads, paramagnetic beads, magnetic beads, glass beads, glass, or glass fiber.

39. The method according to claim 38, wherein, The solid-phase substrate includes a coating, and the coating includes silica, ceramic, polymer, oligonucleotide, or a mixture thereof.

40. The method according to claim 22, wherein, The solid-phase substrate includes a plurality of paramagnetic beads.

41. The method according to claim 22, wherein, The fluid conduit includes a fluid capture region configured to receive the immiscible fluid.

42. The method according to claim 22, wherein, The magnetic field is applied to a region of the capture site that is less than at least one dimension of the capture site.

43. The method according to claim 22, wherein, Flowing the elution buffer through the fluid conduit includes flowing the elution buffer in a direction opposite to the flow direction of the immiscible fluid.

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