Patent Application
20240390906
The isolation of nucleic acid is an important step in many biochemical and diagnostic procedures. For example, the separation of nucleic acids from the complex mixtures in which they are often found is needed before other studies and procedures are conducted, e.g., detection, cloning, sequencing, amplification, hybridization, cDNA synthesis, etc. Methods of isolating nucleic acids from complex starting materials like whole blood, blood serum, urine, respiratory tract secretions, comprise lysis of biological material in the presence of protein inactivating conditions (e.g., protein degrading enzymes), followed by separation of nucleic acids from contaminants by methods including solvent precipitation, solid phase extraction, and dialysis of the nucleic acids. However, the presence of large amounts of cellular or other contaminating material, e.g., proteins, carbohydrates, or salts, in such complex mixtures often impedes many of the reactions and techniques used in molecular biology. Thus, improved methods for isolating and extracting nucleic acids from complex mixtures are needed for diagnosis of microbial infections, forensic science, tissue and blood typing, detection of genetic variations, etc.
This disclosure relates to methods and systems suitable for extracting nucleic acids from a biological sample. In some embodiments, a system for performing PCR is provided. The system includes 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 trapping site disposed along a length of the fluid conduit, the trapping site having a cross-sectional area larger than an average cross-sectional area of the fluid conduit; a plurality of wells disposed on a body of the cartridge, wherein each of the wells are configured to receive one of a plurality of fluids, wherein a portion of the plurality of wells are in fluid communication with the fluid conduit; and one or more magnetic field sources disposed adjacent the trapping site. The system includes a PCR device coupled to the cartridge. The PCR device includes an optical PCR cavity including a light absorbing layer. In some embodiments, each of the plurality of wells are configured to receive a storage container comprising the one of the plurality of fluids, wherein the storage container is configured to be punctured to release the fluid therein. In some embodiments, the plurality of fluids comprises one or more of a lysis buffer, a wash buffer, alcohol, an immiscible fluid, or an elution buffer. In some embodiments, at least one well of the plurality of wells comprises a solid-phase substrate. In some embodiments, the plurality of wells comprise: a first well configured to receive a lysis buffer; a second well configured to receive a first wash buffer; a third well configured to receive a solid-phase substrate; a fourth well configured to receive an immiscible fluid; a fifth well configured to receive a second wash buffer; and a sixth well configured to receive an elution buffer. In some embodiments, the first well and the third well are in fluid communication with the sample chamber. In some embodiments, the second well, fourth well, fifth well, and sixth well are in fluid communication with the fluid conduit. In some embodiments, the cartridge further comprises 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 provide a fluid from the sample chamber or the plurality of wells to the fluid conduit. In some embodiments, a second valve is configured to provide a fluid from the fluid conduit to the PCR device. In some embodiments, the system includes further comprising 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 trapping site has a cross-sectional area at least 10% larger 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 trapping site of the fluid conduit. In some embodiments, a shape of the trapping 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 vary a position, intensity, or field of at least one of the one or more magnetic field sources. In some embodiments, the light absorbing layer comprises a material comprising a light absorbance greater than 50%. In some embodiments, the system includes one or more heaters located adjacent the trapping site. In some embodiments, the one or more heaters are configured to heat components in the trapping site. In some embodiments, the optical PCR cavity is surrounded by external enclosure. In some embodiments, the external enclosure comprises 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 a sample during optical PCR.
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 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 trapping site disposed along a length of the fluid conduit, the trapping site having a cross-sectional area larger than an average cross-sectional area of the fluid conduit; and a plurality of wells disposed on a body of the cartridge, wherein each of the wells are configured to receive one of a plurality of fluids, wherein a portion of the plurality of wells are in fluid communication with the fluid conduit; and one or more magnetic field sources disposed adjacent the trapping site. The method includes a PCR device in communication with the cartridge. The PCR device includes an optical PCR cavity including a light absorbing layer configured for light-to-heat conversion. The method includes providing a biological sample to the inlet of the sample chamber. The method 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. The method includes flowing a solid-phase substrate from a second well of the plurality of wells to the sample chamber to produce a sample fluid including extracted nucleic acids bound to the solid-phase substrate. The method includes flowing the sample fluid from the sample chamber to the fluid conduit. The method includes applying a magnetic field to trap the solid-phase substrate in the sample fluid in the trapping 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 the nucleic acids to the solid-phase substrate. In some embodiments, mixing comprises 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 comprises an alcohol-based wash buffer. In some embodiments, the alcohol-based wash buffer comprises ethanol or propanol. In some embodiments, the method does not include flowing air through the fluid conduit to dry either 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 sufficient force on the solid-phase substrate to keep the solid-phase substrate trapped 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 trapping site has a cross-sectional area at least 10% larger than an average cross-sectional area of the fluid conduit. In some embodiments, the trapping site is characterized by a hexagonal shape. In some embodiments, the system further comprises one or more heaters located adjacent the trapping site. In some embodiments, the method further comprises heating the trapping site using the one or more heaters during at least one of: 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. In some embodiments, the method includes varying the magnetic field to move the solid-phase substrate in the trapping site 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 eluted nucleic acids from the solid-phase substrate are transferred into the elution buffer, wherein flowing the elution buffer comprises flowing the eluted nucleic acids away from the trapping site. In some embodiments, the immiscible fluid comprises one or more of mineral oil, silicone oil, hexadecane, paraffin oil, fluorinerts, fluorinated oils, or mixtures thereof. In some embodiments, the solid-phase substrate comprises a plurality of beads, paramagnetic beads, magnetic beads, glass beads, glass, or glass fibers. In some embodiments, the solid-phase substrate comprises a coating comprising silica, ceramic, polymers, oligonucleotides, or mixtures thereof. In some embodiments, the solid-phase substrate comprises a plurality of paramagnetic beads. In some embodiments, the fluid conduit comprises a fluid trapping region configured to receive the immiscible fluid. In some embodiments, the magnetic field is applied to an area of the trapping site that is smaller than at least one dimension of the trapping site. In some embodiments, flowing the elution buffer through the fluid conduit comprises flowing the elution buffer in a direction opposite to a direction of flow of the immiscible fluid.
Numerous benefits are achieved by way of the present invention over conventional techniques. For example, embodiments of the present invention speed up the overall extraction process, result in higher yields of nucleic acids, and minimize interferences with downstream assays for improved polymerase chain reaction (PCR) amplification. In some embodiments, flowing an immiscible fluid in a fluid conduit following one or more washing steps entirely removes residual washing fluids, including residual contaminants, so that the drying of the fluid conduit is not necessary and it is possible to elute nucleic acids directly after this step. In some embodiments, the fluid conduit for extracting and isolating nucleic acids includes a chamber for trapping the solid-phase substrate in the fluid conduit to increase contact between fluids 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 of sample processing and performing optical PCR. These and other embodiments of the disclosure, along with many of its advantages and features, are described in more detail in conjunction with the text below and corresponding figures.
The disclosure will be readily understood by the following detailed description in conjunction with the accompanying drawings, wherein like reference numerals designate like structural elements, and in which:
In the following detailed description, references are made to the accompanying drawings, which form a part of the description and in which are shown, by way of illustration, specific embodiments in accordance with the described embodiments. Although these embodiments are described in sufficient detail to enable one skilled in the art to practice the described embodiments, it is understood that these examples are not limiting; such that other embodiments may be used, and changes may be made without departing from the spirit and scope of the described embodiments.
Conventional methods of isolating nucleic acids for performing PCR generally require an initial nucleic acid isolation step, to separate the nucleic acid from materials that may interfere in the detection, hybridization, and/or amplification techniques that are used. A range of methods are known for the isolation of nucleic acids, but these rely on a complex series of extraction and washing steps and are time consuming and laborious to perform. For example, methods for isolating nucleic acids from complex starting materials involve lysis of the biological material, possibly in the presence of protein degrading enzymes, followed by several extractions with organic solvents, ethanol precipitation, centrifugations, and dialysis of the nucleic acids. Not only are such methods 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 where several samples are simultaneously processed.
Solid phase extraction is one method of isolating and extracting nucleic acids from a biological sample. During solid phase extraction, nucleic acids in a biological sample bind to a solid-phase substrate. In some embodiments, the solid-phase substrate can be plurality of beads coated with silica or other materials. The solid-phase substrate (e.g., beads) are then washed with alcohol-based or other solutions to remove the supernatant and loosely bound impurities from the nucleic acids bound to the solid-phase substrate. For example, the solid-phase substrate is washed with a low pH wash buffer or an alcohol-based wash buffer to remove any interfering substances before assaying the extracted nucleic acids. As used herein, a “low pH wash buffer” may be a wash buffer having 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 which may interfere with downstream processes, the surface of the solid-phase substrate is dried (e.g., using air) after one or more washing steps. After washing and drying the solid-phase substrate, the nucleic acids are eluted from the solid-phase substrate with a low ionic strength water-based buffer.
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 before assaying the extracted nucleic acids. For example, guanidinium salts used in the lysis buffers are typically removed so as to not interfere with downstream assays. Conventionally, several alcohol-based washes are conducted to remove residual salts from the system. In addition, the alcohol-based wash buffers (e.g., ethanol, propanol, etc.), which are often used in the washing steps, are also removed in order to have high extraction yields and prevent interference with downstream assays. In order to remove the alcohol-based wash buffers, the process typically requires drying the solid-phase substrate, for example, by flowing air over the solid-phase substrate, heating the solid-phase substrate, or applying a negative pressure to the solid-phase substrate. This introduces limitations on the procedure and is often hard to achieve in cartridges, particularly for cartridges that automate sample preparation. For example, high temperatures are sometimes used to evaporate the ethanol; however, high temperatures can degrade nucleic acids, particularly RNA. When air drying the solid-phase substrates, substantial time is required to remove or reduce the alcohol-based wash buffers to acceptable levels (typically less than 1 wt. % in the elute).
Embodiments of the present invention provide methods and systems for performing optical PCR. The methods and systems include eluting nucleic acids from a solid-phase substrate that utilize an immiscible fluid to remove residual reagents or impurities (e.g., alcohol-based wash buffers) from the solid-phase substrate. In some embodiments, the method provides a means to elute nucleic acids bound to surfaces of a solid-phase substrate as part of nucleic acid purification process that uses an immiscible fluid instead of air drying to get rid of residual reagents. In particular 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 speeds the overall extraction process, results in higher yields, and minimizes interferences with downstream assays.
Additionally, specific advantages have been found when using the aforementioned immiscible fluid with a specifically designed fluid conduit for isolating and extracting nucleic acids. Conventional fluid conduits utilize a conduit with a uniform cross-sectional area in which the solid-phase substrate may be trapped in some region of the fluid conduit. In the fluid conduit, a series of lysis, wash, and elution buffers can be passed through the conduit for isolating and extracting the nucleic acids. However, these methods do not provide adequate contact for the lysis, wash, and elution buffers with the solid-phase substrate. Therefore, a plurality of washes are required to achieve the desired isolation and extraction of the nucleic acids. This leads to longer times for the overall extraction process, lower yields of nucleic acids, increased damage to the nucleic acids, and causes more interference with downstream assays. As a result of poor extraction and lower yields of nucleic acids during sample processing, amplification and detection during PCR can be compromised.
In some embodiments, the present disclosure describes a system for performing optical PCR with an improved process for extracting and isolating nucleic acids. In some embodiments, the system comprises a cartridge and a PCR device in communication with the cartridge. The cartridge may include a fluid conduit including a chamber for trapping the solid-phase substrate in the fluid conduit at a trapping site. In some embodiments, the chamber has a larger cross-sectional area than the fluid conduit for receiving and retaining the solid-phase substrate. For example, the solid-phase substrate can be a plurality of paramagnetic beads comprising a silica coating for adhering a biological sample to the solid-phase substrate. The paramagnetic beads can be trapped in the chamber and a magnetic field can be applied to the chamber to retain the paramagnetic beads in the chamber. The magnetic field or magnetic field gradient can be varied in intensity or orientation while maintaining sufficient force to keep the paramagnetic beads trapped while allowing for better mixing with the sample, wash buffer, immiscible fluid, or elution buffer. A wash buffer can be passed through the fluid conduit. As the wash buffer flows through the chamber containing the paramagnetic beads, the magnetic field can be directed in a plurality of directions (e.g., along the X-axis, Y-axis, or Z-axis) with respect 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 absent this increased contact. An immiscible fluid can also be passed 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 contact of the working fluid with the solid-phase substrate to preferentially remove the alcohols, salts, or other interfering substances. In some embodiments, other means of trapping the solid-phase substrate are contemplated, for example, trapping beads by a weir in the conduit.
In some embodiments, the 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. A biological sample can be provided to a sample chamber of the cartridge. The method includes mixing a biological sample with a lysis buffer to produce a first fluid. In some aspects, the lysis buffer comprises guanidinium. In some embodiments, the method includes mixing the first fluid with a binding buffer (e.g., an alcohol-based binding buffer optionally comprising a solid-phase substrate) to produce a second fluid. The second fluid is in contact with a solid-phase substrate (e.g., paramagnetic beads) to bind the nucleic acids to the solid-phase substrate. The solid-phase substrate is subsequently washed with fluids 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 lysed 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 may include an optical PCR cavity including a light absorbing layer configured for light-to-heat 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 illuminate the light absorbing layer of the optical PCR cavity for light-to-heat conversion to perform thermal cycling of the eluted nucleic acids.
These and other embodiments are discussed below with reference to the figures, however, those skilled in the art will readily appreciate that the detailed description given herein with respect to these figures is for explanatory purposes only and should not be construed as limiting.
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, a sample fluid may be provided to the sample chamber 110 via the inlet 112. The inlet 112 can be an injection port for receiving a sample fluid from a collection device (e.g., a hypodermic needle). In some embodiments, a sample fluid may be pre-processed before providing the sample fluid to the sample chamber 110. For example, a whole blood sample can be centrifuged to separate blood cells and other solid components from serum or plasma. The separated serum or plasma can be optionally be diluted or mixed with reagents 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 fluids) to regions of the cartridge 105. In some embodiments, the pump is configured for bubble mixing in the sample chamber.
The cartridge 105 may include a plurality of wells. The plurality of wells may be disposed on the body of the cartridge 105. Each of the plurality of wells may include a concave receptable. The concave receptable may extend into the body of the cartridge 105. The concave receptacle of the plurality of wells is configured to receive a storage container therein. For example, the storage container can be a blister pack that can be received in the well. The blister pack may be configured to be punctured to release a fluid therein. In some embodiments, one or more of the plurality of wells can include a groove (e.g., on the outer circumference of the well). The storage container can include an engagement member (e.g., a prong) to engage with the groove to secure the storage container in the well. For example, the storage container can snap fit into the well.
The cartridge 105 includes one or more wells of the plurality of wells that are in fluid communication with the sample chamber 110. For example, the cartridge 105 includes a first well 120 and a second well 125 that are in fluid communication with the sample chamber 110 via channels. In some embodiments, the first well 120 is configured to receive a storage container including a lysis buffer and the second well 125 is configured to receive a storage container including a solid-phase substrate (e.g., paramagnetic beads). In some embodiments, the lysis buffer comprises guanidinium. The lysis buffer from the first well 120 can be released from the storage container to provide the lysis buffer to the sample chamber 110 via channel 121 to extract nucleic acids from the sample fluid. The solid-phase substrate from the second well 125 can be released from the storage container to provide the solid-phase substrate to the sample chamber 110 via channel 126 to bind the extracted nucleic acids to the solid-phase substrate. Channels 121 and 126 may include valves or actuators to control the flow of fluids to the sample chamber 110. In some embodiments, lysis of the biological sample and binding of the extracted nucleic acids to the solid-phase substrate can be completed in a pre-processing step and then supplied to the sample chamber 110.
The cartridge 105 may include a fluid conduit 130 including a trapping site 135. The fluid conduit 130 includes a trapping site 135 for trapping the solid-phase substrate in the sample fluid in the fluid conduit 130. In some embodiments, the trapping 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 trapping site 135 that may have an enlarged height, width, and/or length compared to the rest of the fluid conduit 130. As discussed herein, a magnetic field source 136 may be located in a region adjacent to or within the trapping site 135. For example, the magnetic field source 136 may be located in a region of the trapping site 135 for trapping or immobilizing the solid-phase substrate. In some embodiments, a controller is coupled to the magnetic field source and is configured to vary the strength, gradient, and/or orientation of the magnetic field source. For example, the controller may be configured to vary a position, intensity, or magnetic field (e.g., width, height, or length of the magnetic field) of at least one of the one or more magnetic field sources. In use, the sample fluid flows through the fluid conduit 130, passing the magnetic field source 136 applied to the trapping site 135, whereupon the solid-phase substrate in the sample fluid is trapped within the magnetic field, while the other components of the sample fluid continue to flow along the fluid conduit 130, removing all the unbound molecules (e.g., supernatant) from the sample fluid. In some embodiments, the trapping site 135 comprises a shallow region (e.g., a weir). In some embodiments, the solid-phase substrate in the sample fluid is trapped in a shallow region of the trapping site 135.
The cartridge 105 includes one or more wells of the plurality of wells that are in fluid communication with the fluid conduit 130. For example, the cartridge 105 can include a third well 140, a fourth well 145, a fifth well 150, and a sixth well 155 that are in fluid communication with the fluid conduit 130 via a plurality of channels. In some embodiments, the third well 140 is configured to receive a storage container including a first wash buffer, the fourth well 145 is configured to receive a storage container including an immiscible fluid, the fifth well 150 is configured to receive a storage container including a second wash buffer, and the sixth well 155 is configured to receive a storage container including an elution buffer.
In some embodiments, the system 100 may include one or more heaters (not shown) adjacent the trapping site 135 of the fluid conduit 130. For example, a plurality of heaters may be disposed below the trapping site 135 of the fluid conduit 130. The one or more heaters may be configured to heat components in the trapping site 135. For example, the one or more heaters can heat a sample fluid that passes the trapping site 135 as it flows through the fluid conduit 130 or the one or more heaters can heat the solid-phase substrate that is trapped in the trapping site 135. In some embodiments, the trapping site 135 can be heated using the one or more heaters 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.
The cartridge 105 may include a first valve 160. The first valve 160 is configured to control the flow of fluids to the fluid conduit 130. In some embodiments, first valve 160 is configured to control of fluids to a first end of the fluid conduit 130. The first valve 160 can be opened to allow the flow of the fluid sample from the sample chamber 110 to the fluid conduit 130. In some embodiments, the pump 108 can pressurize the flow of the sample fluid through the first valve 160 to the fluid conduit 130. For example, the first valve 160 an be opened to flow the sample fluid through the fluid conduit 130 to trap the solid-phase substrate in the trapping site 135. After the solid-phase substrate is trapped in the trapping site 135 of the fluid conduit 130, the first valve 160 can be actuated to allow the first wash buffer from the third well 140 to flow to the fluid conduit 130. The first wash buffer can remove all the unbound molecules (e.g., supernatant) from the sample fluid. The first wash buffer including the unbound molecules can be supplied to a waste chamber 175. In some embodiments, the first valve 160 can be actuated to sequentially flow the immiscible fluid from the fourth well 145 and the second wash buffer from the fifth well 150.
The 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 well 155 to the fluid conduit 130. In some embodiments, the elution buffer from the sixth well 155 can flow counter to the other fluids supplied to the fluid conduit 130. The second valve 165 can also be configured to supply the eluted nucleic acids to the PCR device 180.
The PCR device 180 may include an optical PCR cavity 182 that is configured to receive the eluted nucleic acids from the fluid conduit 130. In some embodiments, the PCR devices described in U.S. Pat. No. 11,406,983 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., which are hereby incorporated by reference, can be used in the system 100. The optical PCR cavity 182 may include a light absorbing layer 184. The light absorbing layer 184 may be configured to, as a function of the absorbed light, elevate a temperature of the light absorbing layer 184 so as to heat the eluted nucleic acids in the optical PCR cavity 182. For example, the light absorbing layer comprises a material having a light absorbance greater than 50% (e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%). The light absorbing layer 184 may comprise one or more layers of metals. 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 absorbing layer is an alloy of any of the aforementioned alloys. The light absorbing material may be a carbon base, non-limiting examples of which include carbon nanotubes, graphite, graphene and/or graphene oxide. The light absorbing layer may comprise of paints such as acrylic paints. The light absorbing layer may comprise a mixture of a metal, metal alloy, carbon base or paint. In some cases, more than one layer of light absorbing materials may be used. One light absorbing layer may absorb light from a light source and transmit light which is not absorbed at first light absorbing layer to a second light absorbing layer to generate heat. In some embodiments, the light absorbing materials 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 primary source of heat for the optical PCR cavity. The light source may be a light-emitting diode (LED), laser diode (LD), tungsten lamp, fluorescent lamp, halogen lamp, mercury lamp, xenon lamp, metal halide lamp or combinations thereof.
In some embodiments, the optical PCR cavity can be surrounded by an external enclosure. The external enclosure may include 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 a sample (e.g., eluted nucleic acids) during optical PCR. In some embodiments, the one or more heaters are configured to heat a sample before or after optical PCR.
The method 1000 includes providing a biological sample to the inlet of the sample chamber (1020). For example, a biological sample can be injected into the inlet of the sample chamber. In some embodiments, the biological sample can be processed prior to being supplied to the sample chamber. For example, plasma or serum can be separated from whole blood and provided to the sample chamber.
The 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). A lysis buffer can be mixed with the biological sample to lyse biological structures such as cells, viruses, organelles or other nucleic acid containing structures in the biological sample to release nucleic acids and supernatant from the structures. In some aspects, the lysis buffer comprises guanidinium. The lysis buffer can lyse cells/organelles in the biological sample and their contents, including nucleic acids, are released into solution. In some embodiments, the biological sample is lysed using any method (e.g., by heating, with ultrasound or a suitable lysis fluid). In some embodiments, the nucleic acids from the lysed biological sample can bind to the solid-phase substrate. The binding may occur in the sample fluid prior to flowing the sample fluid, or at other points in the process or in another liquid medium.
The method 1000 includes flowing a solid-phase substrate from a second well of the plurality of wells to the sample chamber to bind the extracted nucleic acids to the solid-phase substrate (1040). The solid-phase substrate may comprise a plurality of beads. In some embodiments, the beads may be magnetic beads or paramagnetic beads. In some embodiments, the beads may be comprised of silica, polymer, or ceramic with a magnetic or paramagnetic core. In some embodiments, the beads can be functionalized (for example with a silica or silica-like coating, with capture oligonucleotides or with charged polymers) to promote 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 can be directly supplied (e.g., injected) into the sample chamber.
The solid-phase substrate can be mixed with lysed biological sample to produce a sample fluid. In some embodiments, the sample fluid comprises nucleic acids bound to a solid-phase substrate. In this example, the sample fluid comprises 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 be bound to the beads. As known to one of ordinary skill in the art, there are numerous ways to bind nucleic acids in the sample fluid to the solid-phase substrate. The nucleic acids released from the cells can bind to the solid-phase substrate while supernatant does not bind to the solid-phase substrate. The supernatant may comprise cell wall debris, proteins, and other components within the cell. For example, the supernatant can be components within the biological samples that are not nucleic acids.
In some embodiments, the solid-phase substrate can be mixed with the extracted nucleic acids from the biological sample by performing bubble mixing using a pump. The solid-phase substrate and the extracted nucleic acids can be bubble mixed in the sample chamber. In some embodiments, the solid-phase substrate and the extracted nucleic acids can be mixed by advection, turbulent mixing, vortex methods, bubble mixing, micro acoustic streaming, stirring, or other mixing methods. Mixing may be achieved by an active mixing device or passive mixing device.
The method 1000 includes flowing the sample fluid including the solid-phase substrate from the sample chamber to the fluid conduit (1050). For example, a valve can 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 can force the sample fluid from the sample chamber to the fluid conduit. In some embodiments, the sample fluid is provided through the fluid conduit to a trapping site. For example, the method includes flowing a sample fluid comprising the nucleic acids bound to the solid-phase substrate through the fluid conduit to a trapping site. The trapping site may comprise a chamber within the fluid conduit. In the embodiment shown in
The method 1000 includes applying a magnetic field to trap the solid-phase substrate in the chamber of the fluid conduit (1060). For example, when the trapping site comprises a chamber, the solid-phase substrate in the sample fluid can be trapped in the chamber by applying a magnetic field (e.g., localized magnetic field) to the chamber volume. In particular, the sample fluid flows through the fluid conduit, passing the magnetic field applied to the chamber, whereupon the solid-phase substrate is trapped within the magnetic field, while the other components of the sample fluid continue to flow along the fluid conduit, removing all the unbound molecules (e.g., supernatant) from the sample fluid. In some embodiments, the chamber comprises a shallow region (e.g., a weir). In some embodiments, the solid-phase substrate in the sample fluid is trapped in a shallow region of the chamber.
The 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 are passed through the fluid conduit. The wash buffer flows through the fluid conduit passing the localized magnetic field, where the solid-phase substrate is trapped 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 comprises an alcohol. In some embodiments, the wash buffer comprises methanol, ethanol, propanol, butanol, or combinations thereof. The wash buffer removes contaminants (e.g., supernatant, unbound cells, etc.) or residual reagents from the solid-phase substrate or the vicinity of the solid-phase substrate. This wash step may be repeated one or more times to remove all contaminants present on the surface of the solid-phase substrate.
The 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, e.g., 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, an immiscible fluid is flowed through the fluid conduit. The immiscible fluid flows within the fluid conduit and passes the localized magnetic field, where the solid phase substrate, for example, implemented in the form of paramagnetic beads, is trapped within the magnetic field. The flow of 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 alcohols, salts (e.g., from the lysis buffer), or other interfering substances left behind after the wash buffer has passed as described in relation to step 1030. Therefore, the immiscible fluid removes any remaining contaminants from the solid-phase substrate. This step may be repeated one or more times to remove all wash buffer present on the surface of the solid-phase substrate.
The method 1000 includes flowing an elution buffer through the fluid conduit to elute nucleic acids from the solid-phase substrate (1090). The elution buffer flows within the fluid conduit and passes the localized magnetic field, where the solid phase substrate is trapped within the magnetic field. The elution buffer elutes (e.g., releases) the nucleic acids from the surface of the solid phase substrate. In some embodiments, the elution buffer may comprise water. In some embodiments, the elution buffer comprises 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, washing buffer, immiscible fluid, and elution buffer. In some embodiments, the following fluids or subsets of the following fluids are passed through the fluid conduit: sample fluid, air gap, washing buffer, air gap, immiscible fluid, air gap, and elution buffer. In some embodiments, the following fluids or subsets of the following fluids are passed through the fluid conduit: sample fluid, air gap, washing buffer, air gap, immiscible fluid, air gap, and elution buffer. In some embodiments, the following fluid mixtures, which can include solids, are passed through the fluid conduit: solid phase substrate, biological sample, lysing buffer, washing buffer, immiscible fluid, and elution buffer.
In some embodiments, the immiscible fluid passes through the fluid conduit prior to the elution buffer to prevent trace elements of wash buffer (e.g., alcohol) along the fluid conduit and/or the solid phase substrate from mixing with the elution buffer. The immiscible fluid can remove, for example, entirely remove, residual washing fluids from the fluid conduit and/or the solid phase substrate, including residual contaminants remaining in the fluid conduit, so that the drying of the fluid conduit or solid phase substrate is not necessary. Therefore, it is possible to elute nucleic acids directly after flowing the immiscible fluid through the fluid conduit.
Therefore, in some embodiments, the present method does not utilize a drying step following the passing of the washing fluid. This drying step, which is utilized in conventional methods, is time consuming and troublesome because additional processing steps (e.g., heating, vacuum, centrifugation) are utilized.
In some embodiments, method includes heating one or more components in the fluid conduit. For example, the fluid conduit site may include one or more heaters adjacent the trapping site. The heaters may disposed below the trapping site of the fluid conduit 130. In some embodiments, the method includes heating a sample fluid that passes the trapping site as it flows through the fluid conduit. In some embodiments, the method includes heating the trapping 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.
The method 1000 includes transferring the eluted nucleic acids to an PCR device (1095). The PCR device is configured to perform nucleic acid amplification through a polymerase chain reaction. In some embodiments, the PCR device includes an optical PCR cavity. The optical PCR cavity includes a light absorbing layer disposed on a substrate. In some embodiments, the substrate is a transparent substrate. For example, the transparent substrate may allow 50% or greater (e.g., 50% or greater, 60% or greater, 70% or greater, 75% or greater, or 80% or greater) of light to pass through the substrate to the light absorbing layer. The light absorbing layer may be configured to, as a function of the absorbed light, elevate a temperature of the light absorbing layer so as to heat the fluid sample in the optical PCR cavity for nucleic acid amplification. In some embodiments, the light absorbing layer has a light absorbance 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 a wavelength of light (e.g., visible light, UV, infrared, etc.).
The method 1000 includes performing PCR on the eluted nucleic acids in the optical PCR cavity (1096). In some embodiments, the system may include a light source for directing light towards the light absorbing layer of the optical PCR cavity. For example, the light source can be one or light emitting diodes. Alternative light sources may include: a laser diode (LD), tungsten lamp, fluorescent lamp, halogen lamp, mercury lamp, xenon lamp, metal halide lamp, or any combination of the foregoing. The light source can direct light towards the optical PCR cavity for nucleic acid amplification through PCR. In some embodiments, light is illuminated through the substrate to the light absorbing layer. The light absorbing layer is configured to perform light-to-heat conversion for PCR thermal cycling. The absorbed light contributes to the photo thermal heating of the light absorbing layer for the thermal cycling of PCR.
It should be appreciated that the specific steps illustrated in
In some embodiments, the solid-phase substrate 216 can be any suitable substrate that attracts and adheres 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, among others. In some embodiments, the solid-phase substrate 216 comprises a plurality of paramagnetic beads optionally including a coating (e.g., silica or capture oligonucleotides). The solid-phase substrate 216 may be used in systems similar to that outlined here to assist in assembling nucleic acid structures. The solid-phase substrate 216 provides large surface to volume ratios useful in exposing relevant bound chemistry. In some embodiments, the solid-phase substrate 216 can be used to isolate and enrich target cells from a biological sample.
Flowing the immiscible fluid through the fluid conduit, after the washing step and before the elution step, results in the unexpected advantage of reducing or eliminating the process of drying the fluid conduit and/or solid-phase substrate to remove residual washing fluid, which is typically necessary using conventional methods. By eliminating the need for drying the fluid conduit and/or solid-phase substrate result in the unexpected advantage of significantly shortening the duration of the nucleic acid isolation and extraction.
In some embodiments, the chamber 410 comprises a larger cross-sectional area than the fluid conduit 405 to trap the solid-phase substrate therein. For example, the fluid conduit 305 comprises a chamber 410 having a larger cross-sectional area than an average cross-sectional area of the fluid conduit 405. In one example, the chamber 410 of the fluid conduit 405 may 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, compared to the rest of the fluid conduit 405. For example,
In some embodiments, a magnetic field source 415 surrounds a portion of the chamber 410 or the entire chamber 410. For example, the magnetic field source 415 may be located at any location along the length or width of the chamber 410 for trapping or immobilizing the solid-phase substrate. In some embodiments, magnets can be disposed along discrete regions of the chamber 410. A controller can be coupled to the one or more magnets. The controller can be configured to move or oscillate the magnetic field source 415 produced by the one or more magnets. The magnetic field source 415 can be moved in any direction to promote mixing of 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 in 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 in the chamber 410 of the fluid conduit 405. The movement of the magnetic field source 415 to different areas of the chamber 410 while a fluid (e.g., wash buffer or immiscible fluid) passes the chamber 410 promotes mixing and contact with the solid-phase substrate.
An exemplary method of isolating and extracting nucleic acids is provided. The exemplary method utilizes a mineral oil as a final wash before elution when conducting solid-phase extraction with magnetic silica beads.
A 250 μl of a sample containing SARS-COV2 RNA at 100 copies/ml, carrier RNA, and total human RNA1 (1 ng/μl) in a viral transport medium was provided. The sample was 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 were magnetically separated from the supernatant in a microfluidic channel. The beads were then washed twice with 500 μl of a low ionic strength 80% ethanol wash buffer. The beads were then washed with 250 μl of PCR-grade mineral oil. The nucleic acids were eluted with 16 μl of TE buffer and assayed by rt-qPCR. Extractions were performed also in tubes. It was found that, compared to air drying the beads after the final wash, the oil wash reduced the time needed for nucleic acid extraction by more than 5 minutes. Additionally, the eluates had higher output RNA concentrations: an average of 970 copies/ml for the oil versus 360 copies/ml for the air drying.
In some embodiments, the fluid conduit 605 may include a fluid trapping region 650. The fluid trapping region 650 can be adjacent or within the chamber 610. For example, the fluid trapping region 650 can be a region that tapers from the chamber 610 to the fluid conduit 605. In some embodiments, the fluid trapping region 650 is a cavity within the chamber 610. The fluid trapping region 650 is configured to trap or hold an immiscible fluid. The fluid trapping region 650 can be used to trap any immiscible fluid so that it does not block the flow of the elution buffer over the solid-phase substrate as further discussed herein.
The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the described embodiments. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the described embodiments. Thus, the foregoing descriptions of specific embodiments are 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. It will be apparent to one of ordinary skill in the art that many modifications and variations are possible in view of the above teachings.
This application claims priority to and is a continuation of International Patent Application No. PCT/US2023/079975, filed Nov. 16, 2023, which claims the benefit of and priority to U.S. Provisional Application No. 63/384,356, filed Nov. 18, 2022, the entire contents of which are hereby incorporated by reference in their entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63384356 | Nov 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/079975 | Nov 2023 | WO |
| Child | 18757304 | US |
