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WO2020112116A1 - Compositions de réactif - Google Patents

Compositions de réactif Download PDF

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Publication number
WO2020112116A1
WO2020112116A1 PCT/US2018/063085 US2018063085W WO2020112116A1 WO 2020112116 A1 WO2020112116 A1 WO 2020112116A1 US 2018063085 W US2018063085 W US 2018063085W WO 2020112116 A1 WO2020112116 A1 WO 2020112116A1
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WO
WIPO (PCT)
Prior art keywords
reagent
gas
solvent
composition
excipient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2018/063085
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English (en)
Inventor
Daniel CURTHOYS
Viktor Shkolnikov
Alexander Govyadinov
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hewlett Packard Development Co LP
Original Assignee
Hewlett Packard Development Co LP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hewlett Packard Development Co LP filed Critical Hewlett Packard Development Co LP
Priority to PCT/US2018/063085 priority Critical patent/WO2020112116A1/fr
Publication of WO2020112116A1 publication Critical patent/WO2020112116A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/40—Mixers using gas or liquid agitation, e.g. with air supply tubes
    • B01F33/401—Methods
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers

Definitions

  • Reagents are used in a variety of chemical and biological tests and processes. Storing these reagents to assure potency and stability impacts outcomes of the tests. For example, if the reagents degrade and/or react during storage, the concentration of reagent provided will be different from the expected amount due to original concentration and/or byproducts of
  • the degradation and/or reaction products may interfere with the desired test and/or method.
  • Reagents are protected against degradation and/or contamination using a variety of approaches. For example, the use of brown colored jars helps reduce ultraviolet light from degrading materials inside. The storage of reagents at cold temperatures may reduce degradation.
  • Point of Care (POC) testing refers to testing that is conducted where the patient is located. Ideally such testing allows diagnosis and beginning a course of treatment based on the diagnosis in a single visit, rather than having to wait for test results to come back from a lab and/or beginning treatment at risk.
  • a challenge to point of care testing has been reagent composition. Simply put, identifying ways to store reagents without cooling and/or degradation have been challenging and, in many cases, unsuccessful.
  • Microfluidics is the field of using small volumes of fluid to perform testing and/or other operations. Microfluidics facilitate point of care testing and other goals.
  • a microfluidic device such as a“lab on a chip” device which would take a sample, e.g. patient’s blood, and perform a test at the point of care.
  • Using microfluidics in point of care testing reduces the size of test equipment, reduces user impact on method outcomes and reagent storage.
  • FIG. 1 shows an example of a reagent composition consistent with this specification.
  • FIG. 2 shows a flowchart for a method of preparing a reagent composition consistent with this specification.
  • FIG. 3 shows an example of a reagent composition consistent with this specification.
  • FIGS. 4A-D show operation of a reagent composition consistent with this specification.
  • FIG. 5 shows a microfluidic device incorporating a reagent composition of this specification.
  • FIG. 6 shows a microfluidic device incorporating a reagent composition of this specification.
  • FIG. 7 shows a microfluidic device incorporating a reagent composition of this specification.
  • FIG. 8 shows a flowchart for a method of using a reagent composition consistent with this specification.
  • FIG. 9 shows a flowchart for a method of preparing multiple solutions using reagent compositions consistent with this specification.
  • the composition includes the reagent, an excipient, and trapped gas.
  • the trapped gas may be present as bubbles.
  • the trapped gas may be present as dissolved and supersaturated gas in the excipient and/or reagent.
  • a solvent is added, the excipient begins to dissolve.
  • a jet of gas is created which mixes the local liquid and disrupts any boundary layers and/or similar gradients which have accumulated.
  • the jet of gas may move the solid composition in the solvent.
  • the material is saturated and/or supersaturated with gas and the gas forms bubbles adjacent to the surface of the composition which move and disrupt the boundary layer.
  • the excipient includes a sugar (a
  • proteins have a tendency to denature. Denaturing refers to the process of partial and/or total alteration of the native secondary, and/or tertiary, and/or quaternary structures of proteins and/or nucleic acids resulting in a loss of bioactivity. Because proteins have both hydrophilic and hydrophobic domains, when placed on a surface, parts of the protein are attracted to the surface and parts of the protein are repelled by the surface. The result is the protein being disrupted from its conformation.
  • the excipient is selected to participate in a later chemical reaction. For example, if the process being supported is PCR, then bases may serve as both an excipient and a co-reagent.
  • Sugars are also useful in some implementations because they form relatively strong crystals/dried configurations. Sugars have a large drop in free energy when solvated. Sugars are capable of very high concentrations in water based solutions. Sugars may also be cost effective and/or safe compared with a variety of other excipient materials.
  • compositions may be substantially free of a trace component which is only present as a contaminant, even when the trace component is detectable and/or measureable. Accordingly, substantially free may be understood to mean not added except as an incidental contaminant to another component.
  • Standard Temperature and Pressure refers to 0 °C (32 °F) and 1 bar (-0.987 atmospheres). This is the standard currently used by lUPAC, the International Union of Pure and Applied Chemistry. Standard Ambient
  • SATP Temperature and Pressure
  • a“gas” is a material or mixture of materials which are gaseous at SATP. Accordingly, CO2 is a gas even when liquefied by pressure, e.g. above 80 bar.
  • This definition is used in this specification and claims because the functional behavior of the material is that the material functions as a gas when the composition dissolves in the solvent. It is the release of gas or consolidation of trapped gas into bubbles which provides the mixing action to increase location dissolution rates. Accordingly, the storage state of the gas is less important than the behavior of the gas when being released.
  • this specification describes a reagent composition including: a reagent; a solvent-soluble excipient; and gas trapped in the reagent composition.
  • the reagent composition contains more gas than an amount of gas required to saturate the reagent and excipient Standard Ambient Temperature and Pressure (SATP).
  • SATP Standard Ambient Temperature and Pressure
  • this specification also describes a method of preparing a reagent composition.
  • the method includes mixing a reagent and a solvent-soluble excipient which is solid at room temperature and pressure. A mixture of 1 ) the reagent, 2) the solvent-soluble excipient, and 3) gas volumes is cooled such that the gas volumes at above ambient pressure are trapped in the solidified mixture.
  • This specification also describes a microfluidic device including: a port to receive a solvent and a plurality of channels connected to the port.
  • a channel of the plurality of channels contains a reagent composition that includes the reagent, and a solvent-soluble excipient.
  • the reagent composition is supersaturated with gas at Standard Ambient
  • This specification also describes a reagent composition including: 50 to 90 wt. % of the reagent; 1 to 20 wt. % of a solvent-soluble, solid excipient; and 0.001 to 1 wt. % of supersaturated gas trapped in the solid excipient and reagent.
  • This specification also describes a method of using a reagent composition including combining a solvent with a reagent composition, where the reagent composition includes: a reagent, a solvent soluble excipient, and sufficient gas to saturate the reagent composition at SATP.
  • FIG. 1 shows an example of a reagent composition (100) consistent with this specification.
  • the reagent composition (100) includes the reagent (1 10), a solvent-soluble excipient (120), and gas (130) trapped in the composition.
  • the composition contains more gas (130) than an amount of gas (130) required to saturate the reagent (1 10) and excipient (120) at Standard Ambient T emperature and Pressure (SATP).
  • SATP Standard Ambient T emperature and Pressure
  • the reagent (1 10) is the material to be mixed into the solvent in order to be available for some purpose.
  • the reagent (1 10) may be a biological macromolecule.
  • the reagent (1 10) is selected from a group consisting of: a protein; an enzyme; a nucleic acid segment; a primer; a probe, and combinations thereof.
  • the reagent (1 10) may be a biological
  • the reagent (1 10) may be and/or include a base, an acid, a buffer, and/or other chemical species.
  • One consideration for the reagent (1 10) is the stability of the reagent (1 10) when mixed with the excipient (120).
  • the reagent (1 10) may be a lyophilized reagent (100).
  • the reagent (1 10) may include a polymerase, for example, taq polymerase. As described above, the reagent (1 10) may include primers. In an example, the reagent (100) may include nucleotides and/or modified
  • the reagent includes a master mix for PCR.
  • the reagent (100) may include an enzyme, for example, a restriction enzyme.
  • a target is amplified by PCR, cut up with a restriction enzyme, and then shotgun sequenced.
  • the reagent (1 10) may be used for a variety of operations.
  • the reagent (1 10) may be used to lyse a cell.
  • the reagent may be used to stabilize DNA in solution.
  • the reagent may contain ethylenediaminetetracetate (EDTA).
  • the reagent (100) may contain other chelators/stabilizers to act upon a solvent.
  • the reagent (1 10) may include a buffer, for example, a magnesium chloride buffer. Magnesium chloride stabilizes polymerase.
  • the reagent (1 10) may include an acid and/or a base.
  • sodium hydroxide (NaOH) may be a reagent (1 10) in a mixture of sucrose as the excipient (120) and carbon dioxide as the gas (130).
  • a NaOH reagent (1 10) may be added to a melt of the sucrose.
  • the melt may be pressurized to 80 bar in a CO2 atmosphere.
  • the melt may be depressurized to 10 bar and then cooled to ambient to form a reagent composition (100) with trapped NaOH particles and CO2 bubbles in a sucrose matrix.
  • the reagent (1 10) may contain an indicator, for example, ethidium bromide.
  • an ethidium bromide reagent (1 10) allows UV detection of DNA down to about 1 nanogram (ng).
  • the reagent (1 10) may include other indicators.
  • the reagent (1 10) may include DNA, RNA, and/or protein fragments. In an example, these fragments include a probe.
  • the fragments may be modified to enhance detection, for example by bonding a fluorescent and/or phosphorescent group to the fragment.
  • the fragment is modified to have a distinctive bond energy to facilitate identification and/or quantification.
  • the reagent (1 10) may be radioactively and/or fluorescently labelled for detection in DNA sequencers.
  • the excipient (120) provides the structural support to contain the gas (130).
  • the excipient (120) is soluble in the solvent that is going to be used to mix the composition (100).
  • the excipient (120) may be chosen to aid a subsequent reaction. In other examples, the excipient (120) may be chosen so as to not interfere with subsequent reactions, measurements, etc.
  • the excipient (120) includes a saccharide.
  • the excipient (120) may include a monosaccharide, a disaccharide, a polysaccharide, and/or an oligosaccharide.
  • the excipient (120) is a modified saccharide.
  • the excipient (120) includes modification to enhance stabilization of the reagent (1 10). For example, the inclusion of a hydrophobic domain such as an alkyl group, or a positive and/or negative charge may serve to enhance the stabilization of the reagent (1 10).
  • the excipient (120) may include: sucrose, lactose, a polysaccharide, cellulose, microcrystalline cellulose, cellulose ethers, sugar alcohols, xylitol, sorbitol, mannitol, gelatin, polyethylene glycol (PEG), polyvinyl alcohol (PVA) and its derivatives, hydroxypropyl methylcellulose, shellac, corn protein (such as zein), etc.
  • excipient (120) may include a polymer.
  • the polymer is an unsubstituted polyglycol.
  • Polyglycols have controllable melting points based on their chain length. This allows the formation of compositions at temperatures below those used for smaller molecules, such as sugar.
  • Polyglycols are soluble in water base solutions.
  • other water soluble polymers provide a blend of controllable properties, including melt temperatures and solubilities for use in water or other solvent systems.
  • polymers provide increased strength compared with smaller chains (e.g. waxes) and also have tunable properties, including hydrophobicity and melting point, for example, by the inclusion of side chains.
  • a mixture of polyglycol, solvent, and reagent is saturated with CO2 at room temperature (or an elevated temperature, e.g., 37° C) in a pressure chamber, for example at 80 bar. Releasing the pressure on the mixture cools the mixture and flashes off some of the solvent.
  • This approach may produce a loading of approximately 0.05 wt. % of the reagent composition (100) of CO2. This amount of gas (130) had a volume
  • the gas (130) forms 0.05 to 0.5 wt. % of the reagent composition (100).
  • a volume of the reagent composition (100) may release 0.1 to 10 volumes of gas (130) when solvated at SATP.
  • the gas (130) may make up 0.025 to 0.10 wt. % of the reagent composition (100).
  • a volume of the reagent composition (100) may release 0.5 to 2 volumes of gas (130) when solvated SATP.
  • an excipient (120) is selected with a melting temperature above the storage requirements for the reagent composition (100).
  • an excipient (120) may be selected that has a melting point of 33° C.
  • an excipient (120) with a melting point of 50° C may be suitable depending on the expected use conditions.
  • excipients (120) with lower melting points allow incorporation of a wider range of reagents (1 10) without degradation of the reagents (1 10).
  • inclusion of a solvent and/or mixture of solvents may reduce the temperature required to form a solution that can be charged with gas and solidified.
  • PEG-10000 may be used as the excipient (120).
  • PEG-10,000 has a melting point of ⁇ 64 °C.
  • the excipient (120) may be melted and loaded with 9 parts reagent (1 10) to 1 part excipient (120), i.e., 90% loading.
  • the mixture may be pressurized to 80 bar with CO2 to introduce -0.05 wt. % CO2 by weight of the reagent composition (100).
  • PEG provides an example of an excipient (120) where the melting temperature may be adjusted by changing the chain length.
  • the table below shows a series of PEG weights and corresponding melting points. The principle of adjusting the melting point by adjusting the chain length may be readily applied to other excipients (120). Similarly, substituting different materials for the excipient, e.g. PPG for PEG provides additional flexibility.
  • the reagent composition (100) includes an enzyme to breakdown the polyglycols (or other polymers) after they are dissolved. That is, the enzyme degrades the polymer when both are present in a solution. Such an enzyme reduces the viscosity impact of the polyglycols on the resulting solution. In some cases this facilitates downstream reactions, and/or movement through a microfluidic device.
  • One enzyme-excipient pair is dehydrogenase with polyglycols such as polyethylene glycol, polypropylene glycol, and/or polytetramethylene glycol.
  • enzymes such as oxidase, dehydrogenase, and hydrolase may be used.
  • polylactic acid (PLA) and/or polyglycolic acid (PGA) is used as an excipient (120)
  • enzymes such as protease and/or lipase may be used to degrade the excipient (120).
  • a starch excipient (120) may be degraded with amylase, diastases, etc.
  • Protein excipients (120) may be broken up with trypsin, chymotrypsin, pepsin, thermolysin, glutamyl endopeptidase, actinidain, elastase, bromelain, and/or papain.
  • Collagen excipients (120) may be broken up with collagenase.
  • Elastin excipient (120) may be degraded with elastase.
  • Polycaprolactone excipient (120) may be degraded with lipase and/or cutinase.
  • a wide variety of natural and/or artificial polymers may be degraded with a properly selected enzyme and/or mixture of enzymes. Even non polymers can be broken up with enzymes such as invertases for sucrose and/or lactase for lactose.
  • the excipient (120) of the reagent composition (100) is solvent- soluble.
  • the solvent may form the basis of the solution including the reagent (1 10) and excipient (120).
  • the solvent may be water, a water-based solution, and/or an alcohol.
  • the solvent may be a
  • hydrocarbon, and the excipient (120) may be an oil soluble composition.
  • the disclosed approach does not require specific chemical characteristics of the solvent, other than its ability to dissolve the reagent (1 10) and excipient (120).
  • the described approach may operate without chemical reactions.
  • the described approach may operate without additional equipment, such a mechanical agitators and/or heaters. Accordingly, such compositions may be described as“self-mixing” as they provide their own agitation when in contact with the solvent. This does not preclude the use of the compositions (100) with mechanical agitation and/or heating.
  • the reagent composition (100) also contains gas (130).
  • the gas (130) may be in the form of volumes, e.g., bubbles at pressure.
  • the gas (130) may be in the form of liquefied gas, e.g., C02 at about 80 bar or greater.
  • the gas (130) may be present as a supersaturated solid. For example, some compositions of propylene glycol are able to hold approximately 70 wt. % C02. If the composition is saturated at higher pressure, the amount of gas (130) that can be trapped in the solid of the composition (100) may be higher.
  • the gas (130) may be present as phase-separated volumes and/or volumes dissolved in the reagent composition (100).
  • the gas (130) may be stabilized by additional elements of the composition, for example, by a metal oxide, metal hydroxide, and/or metal-organic compound and/or complex.
  • the volumes may be formed by mixing gas (130) into the formulation. Specifically, the volumes may be formed by reducing the pressure on the formulation, causing the excess gas to nucleate and form bubbles in the reagent composition (100). The pressure in the volumes will be related to the pressure on the reagent composition (100) when the reagent composition (100) solidifies and/or becomes viscous enough to entrap the volumes.
  • the gas (130) may be carbon dioxide.
  • the gas (130) is at least 90 % by weight carbon dioxide.
  • Carbon dioxide is able to form a compact phase (liquid, solid) at lower temperatures and pressures (e.g., about 80 bar) than oxygen, nitrogen, and/or other atmospheric gases (130).
  • oxygen may be used to provide an oxygen saturated reagent (1 10) solution after dissolution.
  • nitrogen and/or argon may reduce degradation of the reagent (1 10) during storage.
  • the use of larger gas molecules, e.g., butane may reduce diffusion losses of the gas during storage, increasing shelf-life.
  • gas (130) selection may be determined based on the eventual use of the reagent composition (100).
  • the use of hydrogen as the gas (130) may allow greater amounts of gas (130) to be stored interstitially and/or with a gas storing component, e.g., a metal-organic framework.
  • the gas (130) in volumes may be at pressure of no less than 2 bar.
  • the gas (130) is present as volumes of compressed gas at a pressure from 2 to 200 bar.
  • the pressure is from 10 to 120 bar.
  • the pressure may be at approximatelyl O bar or approximately 80 bar.
  • Higher pressures allow for greater volumes of gas (130) to be released, producing more agitation of the solvent.
  • Higher pressures also imply stronger containment by the reagent composition (100) to retain the gas (130) volume until the material is dissolved.
  • the pressure is selected to remain below the condensation pressure at room temperature.
  • the critical point for carbon dioxide is at approximately 73.8 bar at 31.1 C. Accordingly, a pressure of 80 bar can liquefy or form a supercritical fluid of CO2 during incorporation into the composition (100) depending on the temperature. Liquid carbon dioxide is a good solvent for many lipophilic organic compounds.
  • the gas (130) may be supersaturated in the reagent composition (100).
  • the mixture of reagent (1 10) and excipient (120) may be saturated with gas (130) at elevated pressure and/or temperature and then rapidly cooled and/or pressure decreased (or both by venting) to trap the gas (130) in the reagent composition (100).
  • the gas (130) may be stabilized by an additional material in the composition.
  • metal oxides, hydroxides, and/or metal organic complexes may be used to increase the gas storage capacity of the composition (100). Hydrogen gas may be useful in this regard due to its small radius and ability to fit in a variety of chemical structures. However, upon release hydrogen gas still provides volumes similar to other gases.
  • the gas stabilizer is a Metal Organic Framework (MOF).
  • the gas stabilizer may be a porous coordination polymer. The present disclosure describes the inclusion of a metal-organic complex with gas containment capability into the reagent composition (100) to increase the amount of gas (130) which may be loaded into the reagent composition (100).
  • Such complexes include copper- tetracarboxylate; 4,4-bipyridine; ZIF-300; and SIFSIX-3-Zn.
  • ZIFs Zeolitic Imidazolate Frameworks
  • SiF6 2_ metal cation with hexafluorosilicate
  • SIFSIX MOFs Metal Organic Frameworks
  • the gas (130) may be stabilized using a metal and/or metal hydride.
  • a metal and/or metal hydride For example, hydrogen is able to be stored interstitially in a variety of metal alloys and some of these structures support reversible absorption and desorption of the hydrogen.
  • Metallic hydrides of intermetallic compounds, in the simplest case the ternary system ABxHn, are advantageous because the variation of the elements allows the properties of these hydrides to be tailored.
  • “A” may be a rare earth and/or an alkaline earth metal which tends to form a stable hydride
  • “B” may be a transition metal which forms unstable hydrides.
  • Some ratios of B: A, where x 0.5, 1 , 2, and/or 5 have been found to form hydrides with a hydrogen to metal ratio (n) of up to two.
  • complex hydrides may be able to store relatively large amounts of hydrogen to be released as gas to provide agitation.
  • high surface area materials for example, porous carbon structures, are able to reversibly store hydrogen gas. The inclusion of such materials provides the ability to increase the total gas (130) stored in the reagent composition (100). The inclusion of such materials may also provide additional controls, either chemical selectivity and/or thermal responsiveness to the storage and/or release of the gas (130) from the reagent composition (100).
  • Such materials may provide the ability to store gas (130) and/or precursors for subsequent reactions in the solution which are otherwise hard to stabilize.
  • some MOF structures are able to hold carbon monoxide and/or acetylene for controlled release. This provides additional ways to store difficult to stabilize reagents, including reactive gases, which are otherwise difficult to provide at point of care to facilitate testing.
  • the gas (130) is released by heating the reagent composition (100).
  • the reagent composition (100) may be heated using a resistive heater to release gas (130) trapped in the reagent composition (100). Heating may also increase dissolution rate and reduce viscosities of the resulting solution. Heating may soften the reagent composition (100) allowing the stored gas (130) to be released.
  • the solvent may be an aqueous solvent.
  • a wide variety of water- soluble excipients (120) and reagent (1 10) combinations are available.
  • the solvent may be a non-aqueous solvent.
  • a wax and/or similar material may be used as the excipient (120) and an organic solvent (e.g., acetone, hexane) used.
  • an organic solvent e.g., acetone, hexane
  • liquid carbon dioxide is a good solvent for many lipophilic compounds, making carbon dioxide a useful choice for lipophilic implementations.
  • trapped gas (130) may be applied to a wide variety of chemistries.
  • the solvent may include other reagents.
  • the solvent includes only the solvent, as the goal is to have the reagent composition(s) provide the reagents for the desired reactions.
  • the reagent composition (100) further includes a buffer, for example, to modify the pH of the resulting solution.
  • a buffer for example, to modify the pH of the resulting solution.
  • CO2 as the gas will produce an acidic solution which may be sub optimal for subsequent operations.
  • Providing a buffer and/or a pH modifier may facilitate integrated preparation of the solution for the next operation.
  • FIG. 2 shows a flowchart for a method (200) of preparing a reagent composition (100).
  • the method (200) includes mixing (220) a reagent (1 10) and a solvent-soluble excipient (120) which is solid at room temperature and pressure and cooling (240) under pressure, a mixture of 1 ) the reagent (1 10), 2) the solvent-soluble excipient (120), and 3) gas volumes (130) such that the gas volumes at above ambient pressure are trapped in the solidified mixture.
  • the various operations described may be used in a variety of orders and combinations to produce reagent compositions (100).
  • the reagent composition (100) contains, a reagent (1 10), an excipient (120), and trapped gas (130), where an amount of trapped gas (130) in the composition (100) is more than an amount required to saturate the reagent (1 10) and excipient (120) at standard temperature and pressure.
  • the trapped gas (130) may be present as volumes of pressurized gas, as supersaturated material (e.g., the excipient) formed by saturating the material at temperature and then cooling to trap the gas, and/or stabilized by a stabilizer, such as a polymer, a metal oxide, and/or a metal organic compound.
  • the method (200) may include mixing (220) the reagent (1 10) and excipient (120).
  • the method (200) may (200) further include increasing the pressure of gas (130) in a container containing the mixture of reagent (1 10) and excipient (120).
  • the pressurization may be with air, a selected gas, e.g., carbon dioxide, or a mixture of gases.
  • gas (130) is allowed to be absorbed by the mixture until saturated.
  • the gas (130) is mixed into the mixture of the reagent (1 10) and excipient (120). Physical mixing reduces the diffusion lengths and boundary layer effects, decreasing the time the mixture needs to be held at pressure in order to reach a given concentration of dissolved gas.
  • the size of bubbles which may be suspended in the mixture is a function of viscosity of the mixture.
  • the mixture is cooled (240) under pressure.
  • the pressurized container is vented. This causes a drop in temperature and may flash off any solvent used, if any.
  • the pressurized container is cooled without venting. The pressure of the pressurized container may be reduced to induce formation of volumes (e.g., bubbles) of a given size and then cooled at pressure to trap the volumes. If there are no mechanically entrapped bubbles, these methods may be used to form gas volumes in a saturated mixture.
  • the reagent (1 10) and excipient (120) may be a slurry, with the reagent (1 10) being solid and the excipient (120) being a liquid prior to pressure release and/or cooling.
  • the reagent (1 10) may be a liquid in the mixture of reagent (1 10) and excipient (120) prior to pressure release and/or cooling.
  • the method (200) may include mechanically breaking the solidified mixture into smaller pieces. This has the advantage of increasing the surface area of the composition.
  • the method (200) includes forming the mixture into shapes and/or using a mold.
  • One advantage of this is that reagent (1 10) concentrations may be converted to other measurements, for example, the length of a fixed width, fixed depth, and fixed concentration composition. Accordingly, an instruction may be to add 5 centimeters of the reagent composition (100) to a solution. This may be useful for adding small and/or precise amounts of reagent (1 10) without a high accuracy balance.
  • the heated, pressurized mixture of gas (130), reagent (1 10), and excipient (120) is extruded.
  • the extrusion can include cutting into pieces.
  • the extrusion is allowed to fall and form droplets using the Plateau-Rayleigh instability.
  • the extrusion may be passed through a screen and/or mesh to produce separation.
  • the extrusion may use a die to produce a high surface area cross section, e.g., a plus sign, a star shape, etc.
  • the mixture further includes a colorant and/or dye which allow rapid identification of the particular reagent (1 10) and/or
  • concentrations of a shared reagent are variations of a single color.
  • a molded button and/or other shape may contain a fixed amount of a reagent (1 10), for example, materials to form 1 liter of a standard composition, e.g., 5% dextrose in lactated Ringers solution.
  • a reagent for example, materials to form 1 liter of a standard composition, e.g., 5% dextrose in lactated Ringers solution.
  • the molded shapes may function similar to unit dose detergent formulations, providing a desired amount of something in a pre-measured component. Because such reagent compositions (100) do not contain the solvent, the reagent
  • compositions (100) have lower weight and volume which facilitate transportation and storage.
  • the mixture is formed at a temperature under 90°C, 80°C, 70°C, 60°C, and/or 50°C at all times, during preparation. In a specific example, the mixture has a temperature of under 80°C during preparation.
  • the use of polyglycols with selected molecular weights and melting points may be used to provide a suitable water-soluble excipient with the desired melting point.
  • the inclusion of a small amount of solvent may facilitate preparation of the mixture. Some, most, and/or all of the solvent may be flashed off as part of the cooling process.
  • FIG. 3 shows an example of a reagent composition (300) including:
  • the excipient (120) is a mono or disaccharide.
  • the supersaturated gas comprises carbon dioxide, and supersaturated gas makes up 0.01 to 0.10 wt. % of the composition.
  • the composition may further include a metal-organic gas absorber.
  • FIGS. 4A-D show the operation of a reagent composition (100) consistent with this specification.
  • the figures show the reagent composition (100) composed of reagent (1 10) and excipient (120) with volumes of trapped gas (130). The composition is in contact with a solvent (450).
  • the solvent (450) has just contacted the reagent composition (100).
  • the solvent (450) begins to dissolve the excipient (120) and reagent (1 10).
  • the solvent (450) thins a wall of a volume of gas (130) to the point where the wall fails.
  • the pressurized gas (130) is then released as shown by the arrows.
  • the gas flow induces motion in the solvent (450) which causes mixing and disruption of the boundary layer. Both of these effects may increase dissolution rate.
  • the gas release is strong enough to move the reagent composition (100) within the solvent (450). Such motion increases the dissolution rate.
  • the reagent composition (100) has broken up into a number of smaller pieces and the process is nearly complete.
  • the reagent composition (100) is provided as a large, unitary piece as shown.
  • the reagent composition (100) is mechanically broken up into smaller pieces.
  • the reagent composition (100) may be produced as particles, chunks, etc.
  • the reagent composition (100) may be formed as beads and/or other shapes with greater surface area and interstitial spaces to increase dissolution rates.
  • FIG. 5 shows a microfluidic device (500) incorporating the reagent compositions (100) of this specification.
  • the microfluidic device (500) includes a port (560) to receive the solvent (450).
  • the port (560) feeds the solvent (450) into a manifold (562) which distributes the solvent (450).
  • the manifold (562) feeds a channel (564) which has portions of the reagent composition (100) stored in the channel (564).
  • the manifold (562) also feeds a chamber (566) with reagent composition (100).
  • the solvent (450) mixes with the reagent compositions (100) and forms desired reagents which may be reacted by bringing them together.
  • This example microfluidic device (500) may be provided with sensors, pumps, gates, heaters, etc.
  • the described reagent compositions (100) may be used to facilitate lab-on-a-chip applications.
  • a microfluidic device may include a number of channels (564) and/or chambers (566) which contain reagent compositions (100) that are used to form solutions when combined with a solvent (450).
  • a solvent (450) such as water, deionized water (Dl water), saline, etc.
  • a chamber (566) containing a reagent composition (100) may include a narrowed exit to contain the mixture while mixing.
  • a chamber (566) may include a vent to allow gas released by the composition (100) to escape from the microfluidic device (500).
  • the vent has a hydrophobic surface to impede an aqueous solvent from leaving through the vent.
  • the vent may have a hydrophilic surface for a hydrophobic solvent.
  • the microfluidic device (500) performs PCR.
  • the reagent compositions (100) may include the components of the Master mix, primers, etc. such that the amplification target and solvent are the only materials added to the device.
  • the device (500) may include probes in reagent compositions (100) to interact with the amplified target.
  • the device (500) may include a restriction enzyme and/or other materials to conduct other
  • microfluidic point of care test devices 500
  • Master mix is a mixture of the components needed to perform PCR, including polymerase, bases, and usually a buffer. Master mix may lack primers, which tend to be specific to the amplification target. Master mix lacks the amplification target as formulated.
  • Microfluidic devices (500) may include a resistive heater to increase the dissolution speed of the reagent composition (100). In an example, the heater is also used to melt a thin layer of the reagent composition (100).
  • a lower melting point material may be used between the heater and the reagent composition (100) as an adhesive.
  • the heater is capable of vaporizing a portion of the solvent, e.g., converting water to steam. This may be used to increase the dissolution rate of the composition (100).
  • FIG. 6 shows a microfluidic device (600) incorporating a reagent composition (100) of this specification.
  • the microfluidic device (600) includes a port (560) to receive a solvent and a plurality of channels (564) connected to the port (560).
  • a channel (564) of the plurality of channels (564) contains a reagent composition (100) including the reagent (1 10) and a solvent-soluble excipient (120), wherein the reagent composition (100) is supersaturated with gas (130) at Standard Ambient Temperature and Pressure (SATP).
  • SATP Standard Ambient Temperature and Pressure
  • FIG. 7 shows a microfluidic device (700) incorporating a reagent composition (100) of this specification.
  • the microfluidic device (700) includes a port (560) to receive a solvent; a plurality of channels (564) connected to the port (564), wherein a channel (564) of the plurality of channels (564) contains a reagent composition (100) including: the reagent (1 10) and a solvent-soluble excipient (120), wherein the reagent composition (100) is supersaturated with gas (130) at Standard Ambient Temperature and Pressure (SATP); a vent hole (770) from the channel (564) near the reagent composition (100); and a heater (772), wherein the reagent composition (100) is adhered to a surface of the heater (772).
  • SATP Standard Ambient Temperature and Pressure
  • the vent hole (770) provides an escape route for the gas produced by the reagent composition (100) while dissolving.
  • a surface of the vent hole (770) has a low surface energy, for example, lower than the solvent, to resist solvent exiting through the vent hole (770).
  • an upper surface of the channel is sloped toward the vent hole (770) and/or contains a recess containing the vent hole (770) to help channel the gas toward the vent hole (770).
  • the heater (772) may be a resistive heater (772).
  • a portion of the composition is allowed to melt and resolidify on a surface of the heater (772). This provides an effective way to adhere the regent composition (100) in the device (700) without additional materials.
  • the approach also keeps the composition (100) from moving away from the heater (772) during handling and/or transportation.
  • FIG. 8 shows a flowchart for a method (800) of using a reagent composition (100) including combining (880) a solvent with a reagent composition (100), where the reagent composition (100) includes: a reagent (1 10), a solvent-soluble excipient (1 10), and sufficient gas (130) to saturate the reagent composition (100) at SATP.
  • the method (800) is performed using a microfluidic device. Examples of such devices are shown in FIGS. 5-7.
  • the method (800) may be performed without a device.
  • the solvent and reagent composition (100) may be combined in a flask, beaker, syringe, pipette, and/or other container.
  • the method (800) may further include heating the solvent and reagent composition (100).
  • the heat may be applied using a resistive heater (772).
  • the method (800) may further include not providing agitation to the solvent other than the agitation produced by the gas (130) in the reagent composition (100).
  • the method (800) may also include measuring a length of the reagent composition (100) to determine an amount of reagent (1 10).
  • FIG. 9 shows a flowchart for a method (900) of preparing multiple solutions using reagent compositions (100) consistent with this specification.
  • the method (900) includes providing (990) a solvent to a microfluidic device (600) to form multiple solutions where the solvent is separately combined with multiple reagent compositions (100) and where each reagent composition (100) includes: a reagent (1 10), a solvent-soluble excipient (120), and sufficient gas (130) to saturate the reagent composition (1 10) at Standard Ambient
  • the solvent is provided at a port (560) of the microfluidic device (600).
  • the solvent may be an aqueous solvent.
  • the use of a single solvent to form multiple solutions in a microfluidic device (600) reduces the number of solutions which need to supply to the microfluidic device (600) and the complexity of the associated interface.
  • the use of gas (130) speeds mixing of the solutions and limits the input by a user.
  • a first solution may contain a master mix to perform PCR on a sample. That solution may be processed through the thermal cycles of PCR and then mixed with a second solution containing a reagent for a test, for example, a probe, an indicator, etc.
  • the solvent may be used for other purposes in the microfluidic device (600), for example, the solvent may also be used to move fluid volumes around the microfluidic device (600). Again this limits the number of ports (560) and number solution providing steps to perform the test with the microfluidic device (600). Generally speaking, reducing the number of operations which require user action also reduces opportunities for user error.
  • the method (900) may further include heating a reagent composition (100) with a resistive heater (772) in the microfluidic device (600).
  • the resistive heater (772) is also used to attach the reagent composition (100) to the microfluidic device (600).
  • the heater (772) may be used to soften and/or melt the reagent composition.
  • the resistive heater (772) may be used to increase dissolution rate and/or solubility in the solvent.
  • the heater (772) may be used to expand the gas (130) when the gas (130) forms bubbles.
  • the resistive heater may heat the solution to limit the gas (130) solubility in the solution and increase bubble formation.
  • the resistive heater (772) may be used as part of a pump system to move the prepared to solution(s) to other locations in the microfluidic device (600) for subsequent operations.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

La présente invention concerne, selon un exemple, une composition de réactif. La composition de réactif comprend un réactif, un excipient soluble dans un solvant et un gaz piégé dans la composition de réactif, la composition de réactif contenant plus de gaz qu'une quantité de gaz requise pour saturer le réactif et l'excipient soluble dans un solvant à température et pression ambiantes standard (SATP).
PCT/US2018/063085 2018-11-29 2018-11-29 Compositions de réactif Ceased WO2020112116A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2018/063085 WO2020112116A1 (fr) 2018-11-29 2018-11-29 Compositions de réactif

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2018/063085 WO2020112116A1 (fr) 2018-11-29 2018-11-29 Compositions de réactif

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020127322A1 (en) * 1999-08-03 2002-09-12 Christiaan Bisperink Foaming ingredient and powders containing it
US20030175947A1 (en) * 2001-11-05 2003-09-18 Liu Robin Hui Enhanced mixing in microfluidic devices
WO2006023564A1 (fr) * 2004-08-17 2006-03-02 Kraft Foods Holdings, Inc. Compositions moussantes sans protéines et procédés de fabrication de celles -ci
US7700130B2 (en) * 2002-04-11 2010-04-20 Medimmune Llc Preservation of bioactive materials by spray drying
US20150060272A1 (en) * 2013-03-15 2015-03-05 Nanomix, Inc. Amperometric detection of limulus amebocyte lysate activation by endotoxin and/or 1-3-beta-d-glucan

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020127322A1 (en) * 1999-08-03 2002-09-12 Christiaan Bisperink Foaming ingredient and powders containing it
US20030175947A1 (en) * 2001-11-05 2003-09-18 Liu Robin Hui Enhanced mixing in microfluidic devices
US7700130B2 (en) * 2002-04-11 2010-04-20 Medimmune Llc Preservation of bioactive materials by spray drying
WO2006023564A1 (fr) * 2004-08-17 2006-03-02 Kraft Foods Holdings, Inc. Compositions moussantes sans protéines et procédés de fabrication de celles -ci
US20150060272A1 (en) * 2013-03-15 2015-03-05 Nanomix, Inc. Amperometric detection of limulus amebocyte lysate activation by endotoxin and/or 1-3-beta-d-glucan

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