Use of dried sodium hydroxide to denature double-stranded DNA

Abstract

The present application relates to a method of denaturing double stranded DNA (dsDNA). In some examples, the methods utilize dried sodium hydroxide. In some examples, the method includes: loading dsDNA into a first portion of a cartridge, where a second portion of the cartridge contains dry sodium hydroxide; and mixing the dsDNA with the sodium hydroxide, thereby denaturing the dsDNA.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 476,432, filed on December 21, 2022, and entitled “Using Dried Sodium Hydroxide to Denature Double Stranded DNA,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to methods for denaturing double-stranded DNA. Background Art

[0004] In preparation for sequencing, DNA libraries often need to be denatured into single-stranded molecules. Some current platforms use formamide at elevated temperatures to perform an on-plate denaturation step. Another option is for the user to perform off-plate denaturation of the DNA using 0.1 M sodium hydroxide solution.

[0005] Both strategies are problematic. Formamide is highly toxic and carcinogenic. Therefore, it requires specialized disposal routes, which not only hinders corporate efforts to achieve social responsibility goals, but is also a major cause of consumer complaints. In addition, the denaturation process using formamide requires the cartridge to have a dedicated heating compartment. Furthermore, due to its high viscosity, the mixing of libraries with formamide makes the on-board fluidics system more complex. On the other hand, user-guided denaturation with sodium hydroxide increases the hands-on time of sequencing, is a potential source of human error, and requires consumers to purchase and store additional chemicals and equipment. In addition, the sodium hydroxide in the solution is a strong corrosive. Therefore, new strategies for on-board library denaturation are needed. Summary of the invention

[0006] The examples herein relate to methods, tubes, and cartridges that can be used to denature double-stranded DNA.

[0007] Some examples herein provide a method of denaturing double-stranded DNA (dsDNA), comprising: loading the dsDNA into a first portion of a cartridge, wherein sodium hydroxide is located in a second portion of the cartridge, wherein the sodium hydroxide is dry, and wherein the dsDNA and the dry sodium hydroxide are not in contact with each other when the dsDNA is loaded into the first portion of the cartridge; and mixing the dsDNA with the sodium hydroxide, thereby denaturing the dsDNA.

[0008] In some examples, the method further includes: neutralizing the sodium hydroxide.

[0009] In some examples, the method further includes: hydrating the sodium hydroxide. In some examples, hydrating the sodium hydroxide is performed before mixing the dsDNA with the sodium hydroxide. In some examples, hydrating the sodium hydroxide includes: adding water to the sodium hydroxide in the second portion of the cartridge when the dsDNA and the sodium hydroxide are not in contact with each other. In some examples, the dsDNA is mixed with the sodium hydroxide to hydrate the sodium hydroxide.

[0010] In some examples, the cartridge includes a constriction that temporarily blocks contact between the dsDNA and the sodium hydroxide when the dsDNA is loaded into the first portion of the cartridge. In some examples, suction is used to mix the dsDNA and the sodium hydroxide. In some examples, a syringe pump is used to generate suction.

[0011] In some examples, the cartridge includes an hourglass-shaped reservoir, and the first portion of the cartridge is an upper compartment of the hourglass-shaped reservoir, and the second portion of the cartridge is a lower compartment of the hourglass-shaped reservoir. In some examples, when the dsDNA is loaded into the first portion of the cartridge, a contraction in the hourglass-shaped reservoir temporarily blocks contact between the dsDNA and the sodium hydroxide. In some examples, suction is used to mix the dsDNA with the sodium hydroxide. In some examples, the suction used to mix the dsDNA with the sodium hydroxide hydrates the sodium hydroxide. In some examples, the method further includes: hydrating the sodium hydroxide, and hydrating the sodium hydroxide before mixing the dsDNA with the sodium hydroxide.

[0012] In some examples, the cartridge includes a tube connecting the first portion of the cartridge with the second portion of the cartridge, and the method further includes: transferring the dsDNA to the second portion of the cartridge using the tube. In some examples, transferring the dsDNA to the second portion of the cartridge using the tube, mixing the dsDNA with the sodium hydroxide. In some examples, transferring the dsDNA to the second portion of the cartridge using the tube, hydrating the sodium hydroxide.

[0013] In some examples, the method further includes hydrating sodium hydroxide, wherein the sodium hydroxide is hydrated prior to transferring the dsDNA to the second portion of the cartridge using the tube. In some examples, hydrating the sodium hydroxide includes passing water through the tube to contact the sodium hydroxide and the dsDNA.

[0014] Some examples herein provide a method for denaturing double-stranded DNA (dsDNA), comprising: loading the dsDNA into an upper compartment of a spin tube containing dry sodium hydroxide, thereby denaturing the dsDNA, wherein a lower compartment of the spin tube contains a neutralizing agent; and centrifuging the spin tube to transfer the denatured DNA to the lower compartment of the spin tube, thereby neutralizing the sodium hydroxide.

[0015] In some examples, a timed reaction of between four (4) and six (6) minutes denatures the dsDNA.

[0016] Some examples herein provide a method for denaturing double-stranded DNA (dsDNA), comprising: loading the dsDNA into a tube containing dry sodium hydroxide and a neutralizing agent comprising a time-triggered coating, thereby denaturing the dsDNA; and incubating the tube to release the neutralizing agent from the time-triggered coating, thereby neutralizing the sodium hydroxide.

[0017] In some examples, the time-triggered coating comprises microspheres. In some examples, the release of the neutralizer comprises: releasing microspheres containing the neutralizer.

[0018] Some examples herein provide a method of preparing a cartridge for denaturing double-stranded DNA (dsDNA), comprising: loading sodium hydroxide into a second portion of the cartridge, wherein the sodium hydroxide is in solution, wherein a first portion of the cartridge is configured to receive the dsDNA; and drying the sodium hydroxide within the second portion of the cartridge.

[0019] In some examples, the method further includes: adjusting the temperature during the drying step. In some examples, adjusting the temperature includes: increasing the temperature.

[0020] In some examples, the method further includes applying vacuum drying during the drying step.

[0021] In some examples, the method further includes applying freeze drying during the drying step.

[0022] In some examples, the drying step is performed in the presence of an inert gas.

[0023] In some examples, the solution comprises an active agent. In some examples, the active agent comprises betaine.

[0024] In some examples, the solution includes an inert agent. In some examples, the inert agent includes sodium chloride.

[0025] In some examples, the solution comprises microspheres containing the sodium hydroxide. In some examples, the microspheres include a coating. In some examples, the coating protects the microspheres from any one or more of moisture and carbon dioxide. In some examples, the coating reduces or prevents any static charge on the microspheres. In some examples, the coating is configured to allow triggered release of the sodium hydroxide.

[0026] Some examples herein provide a method of preparing a cartridge for denaturing double-stranded DNA (dsDNA), comprising: drying a solution containing sodium hydroxide on a porous structure; and loading the porous structure into a second portion of the cartridge, wherein the first portion of the cartridge is configured to receive the dsDNA.

[0027] In some examples, the porous structure includes porous glass beads.

[0028] In some examples, the solution comprises an active agent. In some examples, the active agent comprises betaine.

[0029] In some examples, the solution includes an inert agent. In some examples, the inert agent includes sodium chloride.

[0030] In some examples, the method further includes: dispensing a solution containing the sodium hydroxide onto the porous structure.

[0031] In some examples, the method further includes: soaking the porous structure with a solution containing the sodium hydroxide.

[0032] In some examples, drying the solution containing the sodium hydroxide on the porous structure includes: adjusting the temperature. In some examples, adjusting the temperature includes: increasing the temperature.

[0033] In some examples, drying the solution containing sodium hydroxide on the porous structure includes applying vacuum drying.

[0034] In some examples, drying the solution containing sodium hydroxide on the porous structure includes applying freeze drying.

[0035] In some examples, the method further includes: soaking the porous structure in the solution containing sodium hydroxide. In some examples, there are more than one soaking step and more than one drying step. In some examples, the application of more than one soaking step and more than one drying step produces a multi-layer porous structure.

[0036] In some examples, the porous structure includes a coating. In some examples, the coating protects the porous structure from any one or more of moisture and carbon dioxide. In some examples, the coating reduces or prevents any static charge on the porous structure. In some examples, the coating allows for triggered release of the sodium hydroxide.

[0037] Some examples herein provide a rotary tube comprising a first compartment comprising sodium hydroxide; and a second compartment comprising a neutralizing agent.

[0038] In some examples, the first compartment is located above the second compartment in the rotating tube.

[0039] In some examples, the neutralizing agent is any one or more of sulfuric acid, phosphoric acid, carbon dioxide, nitric acid, and hydrochloric acid.

[0040] In some examples, the sodium hydroxide includes dry sodium hydroxide.

[0041] In some examples, the sodium hydroxide is in solution. In some examples, the concentration of the sodium hydroxide in solution is between 0.1N and 1.6N.

[0042] It should be understood that any corresponding features / examples of each of the aspects of the present disclosure as described herein may be implemented together in any appropriate combination, and any features / examples from any one or more of these aspects may be implemented together with any of the features of the other (multiple) aspects as described herein in any appropriate combination to achieve the benefits as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1A Schematically illustrates an embodiment of denaturing a dsDNA library using an hourglass-shaped reservoir, wherein the library is loaded into the upper compartment of the hourglass-shaped reservoir and dry sodium hydroxide is located in the lower compartment of the hourglass-shaped reservoir. After loading, the library is transferred to the bottom portion of the cartridge to allow the denaturation reaction to occur.

[0044] Figure 1B An example of denaturing a dsDNA library is schematically shown, wherein the dsDNA library is loaded into a first compartment of a cartridge, which is connected via a tube to a second compartment of the cartridge containing dry sodium hydroxide. After loading, the dsDNA library is transferred to the compartment containing the dry hydroxide to denature the dsDNA library.

[0045] Figure 1CAn example of denaturing a dsDNA library is schematically shown, where dry sodium hydroxide is located in a tubing system connected to a compartment that the dsDNA library is loaded in. After loading, water flows through and dissolves the sodium hydroxide as it is transferred into the compartment containing the dsDNA library.

[0046] Figure 2A An example of denaturing and neutralizing a dsDNA library is schematically shown, wherein the dsDNA library is loaded into the upper compartment of a transfer tube containing dry sodium hydroxide. A neutralizing agent is contained in the lower compartment of the transfer tube. After denaturing the dsDNA library, centrifugation transfers the dsDNA library to the lower compartment of the transfer tube, where the hydroxide is neutralized.

[0047] Figure 2B Denaturing and neutralizing a library is schematically shown, where a dsDNA library is added to a rotating tube containing dry sodium hydroxide and a neutralizing agent with a time-triggered coating. After a period of time, the dry sodium hydroxide denatures the dsDNA library, and then the neutralizing agent is released, thereby neutralizing the hydroxide.

[0048] Figure 3A The results shown indicate that final sodium hydroxide concentrations between 0.8 N and 1.6 N result in PF% equivalent to the standard workflow using sodium hydroxide, and Figure 3B The GC% on the human genome shows no bias in GC coverage.

[0049] Figure 4 Shown is a protocol for denaturing a library in a single well.

[0050] Figure 5 A scheme for denaturing a library is shown, where two wells are used and the wells are separated by a membrane.

[0051] Figure 6 and Figure 7 Schematic diagram showing the denaturation workflow.

[0052] Fig. 8A A NaOH block is shown in a dry environment, where the NaOH is uncoated. Figure 8B Microspheres containing a neutralizing agent are shown.

[0053] Fig.9A and Fig. 9B Shown is a graphical representation of data showing the delayed timed release of active agent from microspheres coated with Opadry and microspheres coated with a double layer coating of hydroxypropylmethylcellulose (HPMC). Fig.10 Schematic diagram showing comparison of the NovaSeqX denaturation workflow with a passive, simplified denaturation workflow.

[0054] Fig.11A and Fig. 11B An exemplary process of a simplified passive workflow for denaturing a library using tubes containing sodium hydroxide and encapsulated and blocked microspheres is shown. Fig. 11C Data showing comparison of sequencing metrics using the NovaSeq and NovaSeq x workflows with sequencing metrics using a simplified passive workflow are provided.

[0055] Fig.12 An example schematic diagram showing a simplified passive sequencing workflow.

[0056] Fig.13A and Fig. 13B Examples of different microspheres containing different coatings are shown, along with the time taken to rehydrate.

[0057] Fig.14 Data showing the values ​​of sequencing metrics using single-encapsulated microspheres and double-encapsulated microspheres are provided.

[0058] FIG. 15A to FIG. 15E Data showing the key sequencing metrics of DNA denaturation using a passive, simplified workflow versus DNA denaturation by an alternative workflow are provided. FIG. 16A to FIG. 16C Data showing the value of secondary sequencing metrics using a passive, simplified workflow to denature DNA versus denaturing DNA by an alternative workflow are provided. DETAILED DESCRIPTION

[0059] Exemplified herein are methods and compositions that enable the use of precisely measured amounts of solid sodium hydroxide as an alternative on-plate library denaturing agent.

[0060] For example, a cartridge containing dry sodium hydroxide is used as a denaturant. Unlike sodium hydroxide solution, which is a strong corrosive agent, dry sodium hydroxide is non-corrosive. Therefore, it is much safer to use and transport a cartridge containing dry sodium hydroxide than if the cartridge contained sodium hydroxide solution.

[0061] Double-stranded DNA (dsDNA) can be loaded into a cartridge containing dry sodium hydroxide. After loading the dsDNA, the dry sodium hydroxide can be hydrated and mixed with the dsDNA, thereby denaturing the dsDNA. The method can also include: neutralizing the sodium hydroxide. In some examples, the dsDNA is part of a dsDNA library.

[0062] the term

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the terms "comprising" and "including" is not restrictive. The use of the term "having" is not restrictive. As used in this specification, the terms "comprising" and "including" are to be interpreted as having an open-ended meaning, whether in a transitional phrase or in the body of the claims. That is, the above terms should be interpreted synonymously with the phrases "at least having" or "at least including". For example, when used in the context of a process, the term "comprising" means that the process includes at least the listed steps, but may also include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the listed features or components, but may also include additional features or components.

[0064] As used herein, the phrase "double-stranded DNA" refers to two (2) polynucleotide chains connected by hydrogen bonds. The phrase "double-stranded DNA" can be used interchangeably with "dsDNA".

[0065] As used herein, the phrase "GC" refers to guanine-cytosine. As used herein, the phrase "GC%" refers to the percentage of nitrogenous bases in a nucleotide molecule that are guanine or cytosine.

[0066] As used herein, the term "cartridge" refers to any housing or container capable of containing any solid or liquid material. As used herein, a "cartridge" may have a single compartment or portion, or may have more than one (1) compartment or portion.

[0067] As used herein, the phrase "hourglass-shaped reservoir" refers to any housing or container that is shaped like an hourglass and is capable of containing any solid or liquid material. As used herein, an "hourglass-shaped reservoir" may have a single compartment or a single portion, or may have more than one (1) compartment or portion.

[0068] As used herein, the phrase "spin tube" refers to any tube that can be used in a centrifuge.

[0069] As used herein, the term "microsphere" refers to any hollow particle having a round or spherical shape. As used herein, a "microsphere" has a size range of 1 μM to 1,000 μM.

[0070] As used herein, the phrase "vacuum drying" refers to a drying method in which moisture is removed from a substance by placing the substance in an enclosed, airtight container and removing the moisture via a vacuum pump.

[0071] As used herein, the phrase "freeze drying" refers to a drying process in which water is removed from a substance by freezing the substance and then removing ice from the substance resulting from freezing the substance.

[0072] Methods for denaturing double-stranded DNA

[0073] Some examples herein provide a method of denaturing double-stranded DNA (dsDNA), comprising: loading the dsDNA into a first portion of a cartridge, wherein sodium hydroxide is located in a second portion of the cartridge, wherein the sodium hydroxide is dry, and wherein when the dsDNA is loaded into the first portion of the cartridge, the dsDNA and the dry sodium hydroxide are not in contact with each other; and mixing the dsDNA with the sodium hydroxide, thereby denaturing the dsDNA. In some examples, the dsDNA is part of a dsDNA library.

[0074] In some examples, the cartridge includes an hourglass-shaped reservoir, and the first portion of the cartridge is an upper compartment of the hourglass-shaped reservoir, and the second portion of the cartridge is a lower compartment of the hourglass-shaped reservoir. Figure 1A An example of denaturing a dsDNA library using an hourglass-shaped reservoir as a cartridge is schematically shown. Figure 1A The workflow shown shows the dsDNA library loaded into the upper compartment (5) of the hourglass shaped reservoir and the dry sodium hydroxide located in the lower compartment (10) of the hourglass shaped reservoir.

[0075] In some examples, the hourglass-shaped reservoir cartridge contains a constriction that temporarily blocks contact between the dsDNA or the dsDNA library and the sodium hydroxide when the dsDNA is loaded into the first portion of the cartridge. Figure 1A As shown, before the dsDNA library is mixed with the dry sodium hydroxide, the dsDNA library is not in contact with the dry sodium hydroxide due to the constriction (15) of the hourglass-shaped reservoir cartridge. In some examples, the constriction (15) temporarily avoids contact between the dsDNA and the dry sodium hydroxide by surface tension.

[0076] The dsDNA or the dsDNA library is mixed with sodium hydroxide (15) to denature the dsDNA or the dsDNA library. Figure 1A As shown, the dsDNA library is mixed with the dried sodium hydroxide in the lower compartment of the hourglass-shaped reservoir (20). In some examples, suction is used to mix the dsDNA library with the dried sodium hydroxide. In some examples, a syringe pump is used to generate suction.

[0077] In some examples, the two (2) parts of the cartridge are two (2) separate compartments connected via tubing. The dsDNA or dsDNA library can be loaded into the first compartment and the dried sodium hydroxide is located in the second compartment. For example, Figure 1BAn example of denaturing a dsDNA library is schematically shown, wherein the dsDNA library is loaded into a first compartment of a cartridge, which is connected via a tube to a second compartment of the cartridge containing dry sodium hydroxide. After the dsDNA library is loaded into the first compartment (25), the dsDNA library can be transferred to the compartment containing the dry sodium hydroxide (30) via a tube (28). This transfer mixes the dsDNA library with the dry sodium hydroxide (35) and denatures the dsDNA library.

[0078] In some examples, the dsDNA is transferred to the second portion of the cartridge using the tube, the dsDNA is mixed with the sodium hydroxide. In some examples, the dsDNA is transferred to the second portion of the cartridge using the tube, the sodium hydroxide is hydrated. In some examples, the sodium hydroxide is hydrated before the dsDNA is transferred to the second portion of the cartridge using the tube.

[0079] In some examples, the dsDNA library is transferred to the compartment containing the dried sodium hydroxide using a fluidics system.

[0080] In some examples, the cartridge includes a first portion in which the dsDNA or dsDNA library is loaded and connected to a tube containing the dried sodium hydroxide. Figure 1C An example of denaturing a library is schematically shown, where dry sodium hydroxide is located in a tubing system (40) connected to a compartment (45) loaded with the dsDNA library. In some examples, after loading the dsDNA library, water flows through and dissolves the sodium hydroxide (50) as water is transferred to the compartment containing the dsDNA library (55) to initiate the denaturation reaction.

[0081] In some examples, for any of the dsDNA denaturation methods described herein, the dsDNA is denatured at a pH equal to or greater than 8.5, e.g., about 8.5 pH, about 8.6 pH, about 8.7 pH, about 8.8 pH, about 8.9 pH, about 9.0 pH, about 9.1 pH, about 9.2 pH, about 9.3 pH, about 9.4 pH, about 9.5 pH, about 9.6 pH, about 9.7 pH, about 9.8 pH, about 9.9 pH, or about 10.0 pH. In some examples, the dsDNA is denatured at a pH greater than 10.0.

[0082] In some examples, denaturing the dsDNA is performed at a temperature between 20° C. and 22° C., such as about 20° C., about 21° C., or about 22° C. In some examples, denaturing the dsDNA is performed at a temperature below 20° C. In some examples, denaturing the dsDNA is performed at a temperature above 20° C.

[0083] In some examples, any of the dsDNA denaturation methods described herein further include: neutralizing the sodium hydroxide. In some examples, neutralizing the sodium hydroxide includes: using a neutralizing agent. In some examples, the neutralizing agent is any one or more of sulfuric acid, phosphoric acid, carbon dioxide, nitric acid, and hydrochloric acid.

[0084] In some examples, any of the dsDNA denaturation methods described herein further include: hydrating the sodium hydroxide. In some examples, the sodium hydroxide is hydrated in its final position so that the sodium hydroxide is not moved after hydration. In some examples, hydrating the sodium hydroxide is performed before mixing the dsDNA with the sodium hydroxide.

[0085] In some examples, hydrating the sodium hydroxide includes adding water to the sodium hydroxide in the second portion of the cartridge when the dsDNA and the sodium hydroxide are not in contact with each other.

[0086] In some examples, the dsDNA is mixed with the sodium hydroxide to hydrate the sodium hydroxide.

[0087] In some examples, any of the dsDNA denaturation methods further comprises: sequencing the dsDNA. In some examples, the sodium hydroxide is hydrated during sequencing.

[0088] In some examples, any of the dsDNA denaturation methods described herein use suction to mix the dsDNA with the sodium hydroxide. In some examples, the suction used to mix the dsDNA with the sodium hydroxide hydrates the sodium hydroxide.

[0089] Some examples herein provide a method of denaturing dsDNA, comprising: loading the dsDNA into an upper compartment of a transfer tube containing dry sodium hydroxide, thereby denaturing the dsDNA, wherein the lower compartment of the transfer tube contains a neutralizing agent; and centrifuging the transfer tube to transfer the denatured DNA to the lower compartment of the transfer tube, thereby neutralizing the sodium hydroxide. In some examples, the dsDNA is part of a dsDNA library.

[0090] For example, Figure 2A An example of denaturing and neutralizing a dsDNA library is schematically shown, where the dsDNA library is loaded into the upper compartment of a transfer tube containing sodium hydroxide (60). The neutralizing reagent is located in the lower compartment of the transfer tube (65). Figure 2AAs shown, after loading the dsDNA library, the dsDNA library and the dry sodium hydroxide are mixed together (70), and a denaturation reaction denatures the dsDNA library. After the denaturation reaction, the transfer tube (60) is centrifuged and the dsDNA library is transferred to the lower compartment of the transfer tube (72) containing the neutralizer, thereby neutralizing the sodium hydroxide.

[0091] In some examples, a timed reaction of between four (4) minutes and six (6) minutes denatures the dsDNA. In some examples, the timed reaction is about four (4) minutes, about five (5) minutes, or about six (6) minutes. In some examples, the timed reaction is shorter than four (4) minutes. In some examples, the timed reaction is longer than six (6) minutes.

[0092] Some examples herein provide a method of denaturing dsDNA, comprising: loading the dsDNA into a tube containing dry sodium hydroxide and a neutralizing agent comprising a time-triggered coating, thereby denaturing the dsDNA; and incubating the tube to release the neutralizing agent from the time-triggered coating, thereby neutralizing the sodium hydroxide. In some examples, the dsDNA is part of a dsDNA library.

[0093] For example, Figure 2B Denaturing and neutralizing a library is schematically shown, where a dsDNA library is added to a rotating tube containing dry sodium hydroxide and a neutralizing agent with a time-triggered coating (75). These components (80) are added together to initiate a denaturation reaction that denatures the dsDNA library. After incubating the tube, the neutralizing agent is released from the time-triggered coating, thereby neutralizing the sodium hydroxide.

[0094] Figure 2B and Figure 4 An example is shown in which denaturation and neutralization of the library occurs in a single well using sodium hydroxide. The time-triggered coating (shell) encapsulates the neutralizer and delays the release of the neutralizer so that when the library is incubated with the sodium hydroxide, the sodium hydroxide is not immediately neutralized. In some examples, the release of the neutralizer occurs after at least about 1 minute of incubation, such as at least about 90 seconds, at least about 2 minutes, at least about 150 seconds, or at least about 3 minutes.

[0095] In some examples, the delay of release after incubation of the neutralizer is related to the thickness of the shell coating the neutralizer. In some examples, the thicker the shell coating the neutralizer, the longer the duration between the release of the library and the neutralizer after incubation with the sodium hydroxide. Exemplary materials that can constitute the shell coating the neutralizer include, but are not limited to: hydroxypropyl methylcellulose (HPMC), cellulose acetate, polyethylene glycol, PVP-co-PVAc, eudragit (e.g., eudragit RL and eudragit RS), isoleucine, Opadry CA, and polyester (e.g., poly(lactic acid-co-glycolic acid (PLGA) copolymer).

[0096] Figure 5 An example is shown in which denaturation and neutralization of the library occur in two wells separated by a time-delay membrane. In the upper well 120, there are microspheres containing a denaturant (e.g., sodium hydroxide) for denaturing the library. In the lower well 125, there are microspheres containing one or more neutralizing agents.

[0097] In some examples, the any one or more neutralizing agents include any one or more of sulfuric acid, phosphoric acid, carbon dioxide, nitric acid, and hydrochloric acid.

[0098] In some examples, the time-triggered coating comprises microspheres. In some examples, the release of the neutralizer comprises: releasing microspheres containing the neutralizer.

[0099] Method for drying sodium hydroxide

[0100] Some examples herein provide a method of preparing a cartridge for denaturing double-stranded DNA (dsDNA), comprising: loading sodium hydroxide into a second portion of the cartridge, wherein the sodium hydroxide is in solution, wherein a first portion of the cartridge is configured to receive the dsDNA; and drying the sodium hydroxide within the second portion of the cartridge.

[0101] In some examples, the concentration of sodium hydroxide in the solution is between 0.1 N and 1.6 N. In some examples, the concentration of sodium hydroxide in the solution is about 0.1 N, about 0.2 N, about 0.3 N, about 0.4 N, about 0.5 N, about 0.6 N, about 0.7 N, about 0.8 N, about 0.9 N, about 1.0 N, about 1.1 N, about 1.2 N, about 1.3 N, about 1.4 N, about 1.5 N, or about 1.6 N. In some examples, the concentration of sodium hydroxide in the solution is less than 0.1 N. In some examples, the concentration of sodium hydroxide in the solution is greater than 1.6 N.

[0102] In some examples, the method further comprises: adjusting the temperature during the drying step. In some examples, adjusting the temperature comprises: raising the temperature. In some examples, the temperature is raised to between 23°C and 35°C, for example, to about 23°C, to about 24°C, to about 25°C, to about 26°C, to about 27°C, to about 28°C, to about 29°C, to about 30°C, to about 31°C, to about 32°C, to about 33°C, to about 34°C, or to about 35°C. In some examples, the temperature is raised to below 23°C. In some examples, the temperature is raised to above 35°C.

[0103] In some examples, the method further comprises: applying vacuum drying during the drying step. In some examples, the method further comprises: applying freeze drying during the drying step.

[0104] In some examples, the drying step is performed in the presence of an inert gas. In some examples, the inert gas is any one or more of helium, neon, argon, krypton, xenon, and radon.

[0105] In some examples, the solution includes an active agent. In some examples, the active agent includes betaine. In some examples, the solution includes an inert agent. In some examples, the inert agent includes sodium chloride.

[0106] In some examples, the solution comprises microspheres containing the sodium hydroxide. In some examples, the microspheres are made of glass or ceramic plastic. In some examples, the microspheres contain a polymer.

[0107] In some examples, the size of the microspheres ranges between 1 μM and 1,000 μM. In some examples, the size of the microspheres ranges between 1 μM and 100 μM.

[0108] In some examples, the microspheres include a coating. In some examples, the coating is made of a metal, such as, for example, iron, aluminum, magnesium, brass, zinc, or any other metal known in the art.

[0109] In some examples, the coating protects the microspheres from any one or more of moisture and carbon dioxide.

[0110] In some examples, the coating is configured to allow triggered release of the sodium hydroxide.

[0111] Some examples herein provide a method of preparing a cartridge for denaturing double-stranded DNA (dsDNA), comprising: drying a solution containing sodium hydroxide on a porous structure; and loading the porous structure into a second portion of the cartridge, wherein the first portion of the cartridge is configured to receive the dsDNA.

[0112] In some examples, the porous structure comprises porous beads. In some examples, the porous structure comprises porous glass beads. In some examples, the porous structure comprises porous polymer beads.

[0113] In some examples, the pore size of the porous structure is between 1.0 μm and 100 μm.

[0114] In some examples, the method further includes: dispensing a solution containing the sodium hydroxide onto the porous structure.

[0115] In some examples, the method further comprises: soaking the porous structure with a solution containing the sodium hydroxide. In some examples, there are more than one (1) soaking step and more than one (1) drying step. In some examples, the more than one (1) soaking step includes two (2), three (3), four (4), five (5), six (6), seven (7), eight (8), nine (9) or ten (10) soaking steps. In some examples, the more than one (1) soaking step includes more than ten (10) soaking steps. In some examples, the more than one (1) drying step includes two (2), three (3), four (4), five (5), six (6), seven (7), eight (8), nine (9) or ten (10) drying steps. In some examples, the more than one (1) drying step includes more than ten (10) drying steps. In some examples, the application of more than one soaking step and more than one drying step produces a multi-layer porous structure.

[0116] In some examples, the porous structure includes a coating. In some examples, the coating protects the porous structure from any one or more of moisture and carbon dioxide. In some examples, the coating reduces or prevents any static charge on the porous structure. In some examples, the coating allows for triggered release of the sodium hydroxide.

[0117] Transfer tube containing denaturant (e.g. sodium hydroxide) and neutralizer

[0118] Some examples herein provide a rotary tube comprising a first compartment comprising sodium hydroxide and a second compartment comprising a neutralizing agent.

[0119] In some examples, the first compartment is located above the second compartment in the rotating tube. In some examples, the first compartment and the second compartment are adjacent to each other in the rotating tube.

[0120] In some examples, the first compartment is an upper compartment, the second compartment is a lower compartment, and the first compartment and the second compartment are separated by a diaphragm. In some embodiments, the first compartment contains microspheres containing a denaturant for denaturing dsDNA. In some embodiments, the dsDNA library is added to the upper compartment. In some examples, the denaturant is sodium hydroxide. In some examples, the second compartment includes microspheres containing a neutralizer for neutralizing the denaturant. In some examples, the diaphragm is a time-delay diaphragm, which is used to: after the dsDNA library is added to the upper compartment, delay the contact between the microspheres in the lower compartment and the dsDNA library. In some examples, the time-delay diaphragm delays the contact between the microspheres in the lower compartment and the dsDNA for at least 1 minute, at least 90 seconds, at least 2 minutes, at least 150 seconds, or at least 3 minutes.

[0121] In some examples, the denaturant includes sodium hydroxide. In some examples, the denaturant includes sodium chloride. In some examples, the denaturant includes trehalose.

[0122] In some examples, the neutralizing agent is any one or more of sulfuric acid, phosphoric acid, carbon dioxide, nitric acid, and hydrochloric acid.

[0123] In some examples, the sodium hydroxide is dry sodium hydroxide.

[0124] In some examples, the sodium hydroxide is in solution. In some examples, the concentration of sodium hydroxide in the solution is between 0.1N and 1.6N, for example, about 0.1N, about 0.2N, about 0.3N, about 0.4N, about 0.5N, about 0.6N, about 0.7N, about 0.8N, about 0.9N, about 1.0N, about 1.1N, about 1.2N, about 1.3N, about 1.4N, about 1.5N, or about 1.6N. In some examples, the concentration of sodium hydroxide in the solution is less than 0.1N. In some examples, the concentration of sodium hydroxide in the solution is greater than 1.6N.

[0125] In some examples, the sodium hydroxide in the solution is dried in a rotating tube. In some examples, the solution is dried by increasing the temperature so that the temperature is between 23°C and 35°C, for example, the temperature is increased to about 23°C, to about 24°C, to about 25°C, to about 26°C, to about 27°C, to about 28°C, to about 29°C, to about 30°C, to about 31°C, to about 32°C, to about 33°C, to about 34°C, or to about 35°C. In some examples, the temperature is increased to below 23°C. In some examples, the temperature is increased to above 35°C.

[0126] In some examples, the sodium hydroxide is dried by applying a vacuum. In some examples, the sodium hydroxide is dried by applying freeze drying.

[0127] In some examples, the step of drying the sodium hydroxide is performed in the presence of an inert gas. In some examples, the inert gas is any one or more of helium, neon, argon, krypton, xenon, and radon.

[0128] Simplified passive workflow for denaturing dsDNA libraries

[0129] Some examples provided herein relate to denaturing dsDNA using a simplified passive workflow. In some examples, the simplified passive workflow includes: a step of denaturing DNA using a denaturing agent and a step of neutralizing the denaturing agent.

[0130] In some examples, the simplified passive workflow includes a tube containing a first type of microsphere and a second type of microsphere. In some examples, a dsDNA library is added to the tube. In some examples, the first type of microsphere includes a denaturant. In some examples, the denaturant includes any one or more of sodium hydroxide, sodium chloride, and trehalose. In some examples, the second type of microsphere includes a neutralizer (blocking agent). In some examples, the denaturant includes any denaturant disclosed in Table 2.

[0131] In some examples, the second type of microspheres include at least one shell encapsulating the microspheres. In some examples, the at least one shell is composed of any one or more of hydroxypropyl methylcellulose (HPMC), cellulose acetate, polyethylene glycol, PVP-co-PVAc, eudragit (e.g., eudragit RL and eudragit RS), isoleucine, Opadry CA, and polyesters (e.g., lactic acid-co-glycolic acid (PLGA) copolymers). In some examples, the at least one shell includes two shells encapsulating the microspheres. In some examples, the two shells are composed of any one or more of hydroxypropyl methylcellulose (HPMC), cellulose acetate, polyethylene glycol, PVP-co-PVAc, eudragit (e.g., eudragit RL and eudragit RS), isoleucine, Opadry CA, and polyesters (e.g., lactic acid-co-glycolic acid (PLGA) copolymers). In some examples, as disclosed in Table 4, the two shells include shell 1 and shell 2.

[0132] In some examples, the second type of microspheres include a neutralizer. In some examples, the neutralizer includes any one or more of sulfuric acid, phosphoric acid, carbon dioxide, nitric acid, and hydrochloric acid. In some examples, the neutralizer includes a neutralizer disclosed in Table 3 (Neutrablock).

[0133] In some examples, after the dsDNA is added to the tube, at least one or more shells of the second microsphere delay the release of the neutralizer. In some examples, after the dsDNA library is added to the tube, the delay in release of the neutralizer is at least 1 minute, at least 90 seconds, at least 2 minutes, at least 150 seconds, or at least 3 minutes.

[0134] In some examples, the pH in the tube is adjusted during the simplified passive workflow. In some examples, during the step of denaturing the dsDNA library, the pH is between about 6 and about 9, e.g., the pH is about 7, or the pH is about 8. In some examples, during the step of neutralizing the denaturant, the pH is between about 12 and 14, e.g., the pH is about 14.

[0135] Working Example

[0136] The following examples are intended to be illustrative only and not limiting of the present disclosure.

[0137] Example 1. Dry sodium hydroxide can denature dsDNA

[0138] A proof-of-concept experiment was performed to show the feasibility of denaturing dsDNA libraries using different concentrations of dry sodium hydroxide.

[0139] Sodium hydroxide solutions of different concentrations were dried overnight in a volume of 5 μl (the different concentrations of sodium hydroxide used are shown in Table 1). The next day, the dsDNA library was directly used to hydrate the sodium hydroxide and initiate the denaturation reaction. The library was read for a single 150-cycle read on a HiSeq X sequencer (Illumina) for sequencing.

[0140] The data show that a wide range of sodium hydroxide concentrations from 0.8N to 1.6N results in PF% being equivalent to the standard workflow using sodium hydroxide solution without any impact on the error rate (see Figure 3A ). In addition, analysis of GC% coverage on the human genome showed no GC coverage bias (see Figure 3B ).

[0141] Table 1.

[0142] Lanes condition Is it dry? 1 standard no 2 0.1N yes 3 0.2N yes 4 0.4N yes 5 0.6N yes 6 0.8N yes 7 1.0N yes 8 1.6N yes

[0143] Example 2. Denaturation Workflow

[0144] Figure 6 A simplified passive denaturation workflow is shown. The user adds the library to a tube and then denatures the library. The denaturant used to denature the library is then blocked. The library can then be sequenced using conventional sequencing methods. Figure 7 Passive library denaturation and no neutralization / blocking workflow are shown. In step 0, the user adds the library to the tube. The library is contacted with block 1 (e.g., microspheres containing sodium hydroxide). Then, a trigger is generated that causes a delay in neutralization of the sodium hydroxide. As described below, this trigger that causes a delay in neutralization can be a time-triggered coating or a time-delayed membrane that encapsulates the microspheres.

[0145] There are at least two ways to carry out this process in a tube. Figure 4 A first embodiment is shown. In this example, a library is added to a tube containing a single well. In the tube, the library is contacted with sodium hydroxide, which denatures the library. Then, a neutralizer is released to neutralize the sodium hydroxide. The neutralizer is contained in a microsphere containing a time-triggered coating. Therefore, there is a delay between the time when the library begins to be incubated in the tube and the release of the neutralizer. The delay can be any length of time between 1 minute and 3 minutes. Figure 5 A second embodiment is shown. In this example, the library is added to a tube containing two wells, and these wells are separated by a time-delay diaphragm. Initially, the library contacts the microspheres containing sodium hydroxide in the upper well 120. Due to the presence of the time-delay diaphragm, after a delay (between 1 and 3 minutes), the sodium hydroxide is neutralized.

[0146] Fig. 8A and Figure 8B An example of a reagent formulation used in a simplified passive denaturation workflow is shown. Fig. 8A As shown, when the sodium hydroxide block is in a tube in a dry environment, the block does not need to be coated. Figure 8B As shown, the microspheres contain a blocking agent (Neutralblock). Examples of blocking agents include:

[0147] 1.334nM Blocker Oligonucleotide

[0148] 1M MOPS

[0149] 10 mM MgCl2

[0150] 17.4% trehalose

[0151] 0.2% Polysorbate 20

[0152] Fig.9A and Fig. 9B Data for delayed 2 minute release of microspheres single coated with cellulose acetate and microspheres double coated with cellulose acetate and HPMC are shown. The data show that the single coated and double coated microspheres delayed release of the neutralizing agent relative to uncoated control microspheres.

[0153] Lyophilized formulations for simplified passive denaturation

[0154] Table 2 provides examples of denaturants that can be used in the simplified passive workflow.

[0155] Table 2. Denaturants

[0156]

[0157] Table 3 provides examples of blocking formulations (neutralizing formulations) (Neutrablock).

[0158] Table 3. Neutrablock

[0159]

[0160] Table 4 provides embodiments of shell formulations that can be used to encapsulate microspheres.

[0161] Table 4.

[0162] Encapsulation ％ Components (+Efka and Makkon) Shell 1 20 Thymol Blue, Cellulose Acetate 50K Shell 2 27 HPMC E5, magnesium stearate (1.23%)

[0163] Simplified passive workflow compared to typical denaturation process

[0164] As shown in Table 5 and Fig.10 As shown, use of a simplified passive denaturation workflow can result in a simplified user experience relative to a typical denaturation process.

[0165] Table 5.

[0166] Typical denaturation process Simplified passive transformation Touchpoints 14 5 TAT(minutes) 15 5 HOT(minutes) 5 <0.5 Partial Count 6 1

[0167] Adjust pH in a simplified passive workflow

[0168] By adjusting the pH from 14 to 7, a sequencing-ready and compatible denatured library was generated. Fig.12 Schematic diagram showing a simplified passive workflow in which the pH is adjusted at steps at different times. When the library is added to the tube (step 0), the pH is between 7 and 8. When the library is in contact with the sodium hydroxide microspheres (step 1) and during denaturation (step 2), the pH is 14. When the neutralizer is released (step 3), the pH drops back to between 7 and 8. Fig.11A and Fig. 11B Schematic diagram showing the addition of libraries to tubes as part of a simplified passive workflow. Fig.11A and Fig. 11B As shown, based on the use of thymol blue, the pH changes from 14 (when the library comes into contact with the sodium hydroxide block and when the library is denatured) to 7 when the sodium hydroxide is neutralized. Fig. 11CComparative data using secondary sequencing metrics in the above workflows and in typical NovaSeq and NovaSeq x workflows are provided. Fig.13A Examples of two different microspheres containing different coatings are shown, along with the time taken to rehydrate. Fig. 13B Examples of microspheres coated with different percentages of cellulose acetate are shown, along with the time it took to rehydrate.

[0169] Comparison of single- and double-encapsulated sequencing metrics

[0170] In a simplified passive workflow, sequencing metrics were compared between single- and double-encapsulated microspheres. Fig.14 As shown, the double-encapsulated microspheres include a first outer layer (shell) of 30% cellulose acetate and a second outer layer (shell) of 20% HPMC, while the single-encapsulated microspheres include an outer layer (shell) of 30% HPMC. Fig.14 The data in show that single encapsulated microspheres have less desirable primary sequencing metric values ​​compared to dual encapsulated microspheres.

[0171] Analyze primary and secondary sequenced metrics using a streamlined, passive workflow

[0172] FIG. 15A to FIG. 15E Data showing comparison of key sequencing metric values ​​in sequencing libraries using simplified passive workflows to denature DNA ("Simplified Workflow" and "Coat Vortex Mixing") versus workflows in which DNA is denatured by alternative workflows ("NovaSeq Workflow" and "NovaSeq x Workflow") are provided. FIG. 16A to FIG. 16C Data showing comparison of error rates in sequencing libraries using a simplified passive workflow to denature DNA ("Coat vortex mixing") with those using workflows to denature DNA by alternative workflows ("Dale std" and "Blocker std denaturation") are provided.

[0173] Additional Notes

[0174] Although various illustrative examples have been described above, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the present disclosure. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the embodiments provided herein.

[0175] It should be understood that any corresponding features / examples of each of the aspects of the present disclosure as described herein may be implemented together in any appropriate combination, and any features / examples from any one or more of these aspects may be implemented together with any of the features of the other (multiple) aspects as described herein in any appropriate combination to achieve the benefits as described herein.

Claims

1. A method for denaturing double-stranded DNA (dsDNA), comprising: loading the dsDNA into a first portion of the cartridge, wherein sodium hydroxide is located in a second portion of the cartridge, wherein the sodium hydroxide is in a dry form, and wherein when the dsDNA is loaded into the first portion of the cartridge, the dsDNA and the dry sodium hydroxide are not in contact with each other; as well as The dsDNA is mixed with the sodium hydroxide, thereby denaturing the dsDNA.

2. The method according to claim 1, further comprising: The sodium hydroxide is neutralized.

3. The method according to claim 1, further comprising: The sodium hydroxide is hydrated.

4. The method of claim 3, wherein hydrating the sodium hydroxide is performed prior to mixing the dsDNA with the sodium hydroxide.

5. The method of claim 4, wherein hydrating the sodium hydroxide comprises: When the dsDNA and the sodium hydroxide are not in contact with each other, water is added to the sodium hydroxide in the second portion of the cartridge.

6. The method of claim 3, wherein the dsDNA is mixed with the sodium hydroxide to hydrate the sodium hydroxide.

7. The method of claim 1, wherein the cartridge comprises a constriction that temporarily blocks contact between the dsDNA and the sodium hydroxide when the dsDNA is loaded into the first portion of the cartridge.

8. The method of claim 7, wherein the dsDNA is mixed with the sodium hydroxide using suction.

9. The method of claim 8, wherein a syringe pump is used to produce the suction.

10. The method of claim 1, wherein the cartridge comprises an hourglass-shaped reservoir, and wherein the first portion of the cartridge is an upper compartment of the hourglass-shaped reservoir and the second portion of the cartridge is a lower compartment of the hourglass-shaped reservoir.

11. The method of claim 10, wherein when the dsDNA is loaded into the first portion of the cartridge, a constriction in the hourglass-shaped reservoir temporarily prevents contact between the dsDNA and the sodium hydroxide.

12. The method of claim 11, wherein the dsDNA is mixed with the sodium hydroxide using suction.

13. The method of claim 12, wherein the pumping used to mix the dsDNA with the sodium hydroxide hydrates the sodium hydroxide.

14. The method according to claim 10, further comprising: The sodium hydroxide is hydrated, wherein the sodium hydroxide is hydrated prior to mixing the dsDNA with the sodium hydroxide.

15. The method of claim 1, wherein the cartridge includes a tube connecting the first portion of the cartridge to the second portion of the cartridge, the method further comprising: The tube was used to transfer the dsDNA to the second portion of the cartridge.

16. The method of claim 15, wherein the dsDNA is transferred to a second portion of the cartridge using the tube and the dsDNA is mixed with the sodium hydroxide.

17. The method of claim 15, wherein the dsDNA is transferred to a second portion of the cartridge using the tube to hydrate the sodium hydroxide.

18. The method according to claim 15, further comprising: The sodium hydroxide is hydrated, wherein the sodium hydroxide is hydrated prior to using the tube to transfer the dsDNA to the second portion of the cartridge.

19. The method of claim 18, wherein hydrating the sodium hydroxide comprises: Water was passed through the tube to contact the sodium hydroxide and the dsDNA.

20. A method for denaturing double-stranded DNA (dsDNA), comprising: The dsDNA is denatured by loading it into the upper compartment of a transfer tube containing dry sodium hydroxide, wherein the lower compartment of the rotating tube contains a neutralizing agent; as well as The spin tube was centrifuged to transfer the denatured DNA to the lower compartment of the spin tube, thereby neutralizing the sodium hydroxide.

21. The method of claim 20, wherein a timed reaction of between four (4) and six (6) minutes denatures the dsDNA.

22. A method for denaturing double-stranded DNA (dsDNA), comprising: loading the dsDNA into a tube containing dry sodium hydroxide and a neutralizing agent comprising a time-triggered coating, thereby denaturing the dsDNA; as well as The tubes are incubated to allow the neutralizing agent to be released from the time-triggered coating, thereby neutralizing the sodium hydroxide.

23. The method of claim 22, wherein the time-triggered coating comprises microspheres.

24. The method of claim 23, wherein the release of the neutralizing agent comprises: The microspheres containing the neutralizing agent are released.

25. A method of preparing a cartridge for denaturing double-stranded DNA (dsDNA), comprising: loading sodium hydroxide into the second portion of the cartridge, wherein the sodium hydroxide is in solution, wherein the first portion of the cartridge is configured to receive dsDNA; as well as The sodium hydroxide within the second portion of the cartridge is dried.

26. The method according to claim 25, further comprising: The temperature was regulated during the drying step.

27. The method of claim 26, wherein adjusting the temperature comprises: The temperature is increased.

28. The method of claim 25, further comprising: Vacuum drying was applied during the drying step.

29. The method of claim 25, further comprising: Freeze drying was applied during the drying step.

30. The method of claim 25, wherein the drying step is performed in the presence of an inert gas.

31. The method of claim 25, wherein the solution comprises an active agent.

32. The method of claim 31 , wherein the active agent comprises betaine.

33. The method of claim 25, wherein the solution comprises an inert agent.

34. The method of claim 33, wherein the inert agent comprises sodium chloride.

35. The method of claim 25, wherein the solution comprises microspheres containing the sodium hydroxide.

36. The method of claim 35, wherein the microspheres comprise a coating.

37. The method of claim 36, wherein the coating protects the microspheres from any one or more of moisture and carbon dioxide.

38. The method of claim 36, wherein the coating reduces or prevents any static charge on the microspheres.

39. The method of claim 36, wherein the coating is configured to allow triggered release of the sodium hydroxide.

40. A method of preparing a cartridge for denaturing double-stranded DNA (dsDNA), comprising: drying a solution containing sodium hydroxide on the porous structure; as well as loading the porous structure into the second portion of the cartridge, The first portion of the cartridge is configured to receive dsDNA.

41. The method of claim 40, wherein the porous structure comprises porous glass beads.

42. The method of claim 40, wherein the solution comprises an active agent.

43. The method of claim 42, wherein the active agent comprises betaine.

44. The method of claim 40, wherein the solution comprises an inert agent.

45. The method of claim 44, wherein the inert agent comprises sodium chloride.

46. ​​The method of claim 40, further comprising: A solution containing the sodium hydroxide is dispensed onto the porous structure.

47. The method of claim 40, further comprising: The porous structure is soaked with the solution containing the sodium hydroxide.

48. The method of claim 40, wherein drying the solution containing the sodium hydroxide on the porous structure comprises: The temperature is adjusted.

49. The method of claim 48, wherein adjusting the temperature comprises: The temperature is increased.

50. The method of claim 40, wherein drying the solution containing sodium hydroxide on the porous structure comprises: Apply vacuum drying.

51. The method of claim 40, wherein drying the solution containing sodium hydroxide on the porous structure comprises: Apply freeze drying.

52. The method of claim 40, further comprising: The porous structure is immersed in the solution containing sodium hydroxide.

53. The method of claim 52, wherein there is more than one soaking step and more than one drying step.

54. The method of claim 53, wherein application of more than one soaking step and more than one drying step produces a multi-layered porous structure.

55. The method of claim 40, wherein the porous structure comprises a coating.

56. The method of claim 55, wherein the coating protects the porous structure from any one or more of moisture and carbon dioxide.

57. The method of claim 55, wherein the coating reduces or prevents any static charge on the porous structure.

58. The method of claim 55, wherein the coating allows for triggered release of the sodium hydroxide.

59. A transfer tube comprising: a first compartment containing sodium hydroxide; and A second compartment contains a neutralizing agent.

60. The transfer tube of claim 59, wherein the first compartment is located above the second compartment in the transfer tube.

61. The rotary tube of claim 59, wherein the neutralizing agent is any one or more of sulfuric acid, phosphoric acid, carbon dioxide, nitric acid, and hydrochloric acid.

62. The rotary tube of claim 59, wherein the sodium hydroxide comprises dry sodium hydroxide.

63. The rotary tube of claim 59, wherein the sodium hydroxide is in solution.

64. The rotary tube of claim 63, wherein the concentration of sodium hydroxide in the solution is between 0.1 N and 1.6 N.

Images