Patent Application
20220314215
In biomedical, chemical, and environmental testing, isolating a component of interest from a sample fluid can be useful. Such separations can permit analysis or amplification of a component of interest. As the quantity of available assays for components increases, so does the demand for the ability to isolate components of interest from sample fluids.
In biological assays, a biological component can be intermixed with other components in a biological sample that can interfere with subsequent analysis. As used herein, the term “biological component” can refer to materials of various types, including proteins, cells, cell nuclei, nucleic acids, bacteria, viruses, or the like, that can be present in a biological sample. A “biological sample” can refer to a fluid or a dried or lyophilized material obtained for analysis from a living or deceased organism. Isolating the biological component from other components of the biological sample can permit subsequent analysis without interference and can increase an accuracy of the subsequent analysis. In addition, isolating a biological component from other components in a biological sample can permit analysis of the biological component that would not be possible if the biological component remained in the biological sample. Many of the current isolation techniques can include repeatedly dispersing and re-aggregating samples. The repeated dispersing and re-aggregating can result in a loss of a quantity of the biological component. Furthermore, isolating a biological component with some of these techniques can be complex, time consuming, and labor intensive and can also result in less than maximum yields of the isolated biological component.
In accordance with examples of the present disclosure, a vertically layered fluid column includes three fluids positioned in fluid layers. Two of the fluids are positioned along a density-differential interface and two of the fluids are positioned along a capillary force-supported interface. In one example, one of the fluids can be positioned along the density-differential interface and can also be positioned along the capillary force-supported interface, e.g., three fluid fluids positioned as layers with a middle layer including two interfaces (one along an upper side and one along a bottom side of the fluid layer). In another example, one of the fluids can be an oil and is positioned along a capillary force-supported interface. In another example, vertically layered fluid column may include four fluids positioned as fluid layers. In this example, two of the fluids can be positioned along the density-differential interface and the two of the fluids can be positioned along the capillary force-supported interface. In further detail, one of the fluids can be a gas, e.g., air, and one of the fluids can be an oil, e.g., mineral oil, and the gas and the oil can be positioned along the capillary force-supported interface. In another example, a biological sample can be included having a biological component that can pass through the density-differential interface and the capillary force-supported interface. In one example, the capillary force-supported interface can separate a lower fluid from an upper fluid, wherein the lower fluid is less dense than the upper fluid. The capillary force-supported interface can be, for example, contained within a fluidic channel having a cross-sectional dimension that is aligned with the capillary force-supported interface is less than 2 mm.
In another example, a biological component processing system includes a vertically layered fluid column including three fluids positioned in fluid layers, wherein two of the fluids are positioned along a density-differential interface, and wherein two of the fluids are positioned along a capillary force-supported interface. The biological component processing system also includes a particulate substrate to pass through the density-differential interface and the capillary force-supported interface. The particulate substrate can include magnetizing particles, for example. In this example, the vertically layered fluid column can be spatially positioned adjacent to a magnet to provide a magnetic field, e.g., to move the magnetizing particles along a z-axis through the density-differential interface and through the capillary force-supported interface. In another example, the particulate substrate can include microparticles that have a density greater than the three fluids positioned in fluid layers, wherein the density of the microparticles is sufficient to allow the microparticles to pass through the density-differential interface and the capillary force-supported interface either by the force of gravity or centrifugation. The particulate substrate can include particle surfaces that are associated with a biological component. A biological sample can also be included, where the particulate substrate includes particle surfaces that are surface-activated to preferentially bind with a biological component relative to secondary components in the biological fluid sample.
In another example, a method of processing a biological component from a biological sample includes loading a biological sample into a vertically layered fluid column including a plurality of fluids positioned in fluid layers, a density-differential interface along which two fluids from the plurality of fluids are positioned, and a capillary force-supported interface along which two fluids from the plurality of the fluids are positioned. The method in this example also includes passing a biological component of the biological sample through the density-differential interface and the capillary force-supported interface.
It is noted that when discussing examples of vertically layered fluid columns, biological component processing systems, or methods of processing a biological component from a biological sample, such discussions can be considered applicable to one another whether or not they are explicitly discussed in the context of that example. Thus, for example, when discussing a multi-fluid density gradient portion in a vertically layered fluid column, such disclosure is also relevant to and directly supported in the context of a microfluidic biological component processing system, or a method of processing a biological component from a biological sample, and vice versa.
Terms used herein will have the ordinary meaning in the relevant technical field unless specified otherwise. In some instances, there are terms defined more specifically throughout the specification or included at the end of the present specification, and thus, these terms can have a meaning as described herein.
With respect more specifically to the vertically layered fluid columns of the present disclosure, as shown in
As an initial matter, the terms “density gradient” or “multi-fluid density gradient” can be used in various contexts herein but refer to the ability of multiple fluids to remain separated in layers due to their density difference (with denser fluids being positioned vertically lower along the column). Thus, there can be multiple fluids that are phase separated, but are still in direct contact at a fluid interface, referred to herein as a “density-differential interface,” which is descriptive of the interface being present as a result of the density difference.
The terms “capillary force” or “capillary force-supported gradient” refer to fluid interfaces that are not provided by their increasing density and their density difference, but rather, the fluids of immediately adjacent layers can have different densities, but less dense fluids can be positioned below denser fluids, and the reason these less dense fluid do not migrate upward is because they are constrained within a fluidic channel due to the surface tension of the fluids at the fluid interface and the interaction of the fluids with walls of this channel portion of the vessel, namely at the “capillary force-supported interface.”
Furthermore, in referring to the figures, as there are several fluids often being described, they may be referred to as a “first,” “second,” “third,” etc., fluid so that they can be described relative to one another and for clarity in describing for understanding the disclosure, but should not be considered to be limiting. For example, fluid 180 could be referred to as the “first fluid,” fluid 170 could be referred to as the “second fluid,” and fluid 160 could be referred to as the “third fluid” or the “fourth fluid,” etc., without consequence to the scope of the disclosure. The mentioning of “first,” second,” “third,” etc., should be viewed in the context of the other layers in the immediately described vertically layered fluid column, biological component processing system, or method of process a biological component, and not confused with other instances where the terms “first,” “second,” “third,” etc., may be used differently in another context. For example, when there are more than three or so fluids, and when there are multiple types of the various fluid interfaces, it may make sense to dispense with the use of “first,” “second,” “third,” etc., with the understanding that these naming conventions can be assigned inferentially based on the spatial relationships from layer to layer.
Referring again to
Example density differences of the first fluid relative to the second fluid (or any two fluids along the multi-fluid density gradient portion) can be from 50 mg/mL to 3 g/mL, from 100 mg/mL to 3 g/mL, from 500 mg/mL to 3 g/mL or from 1 g/mL to 3 g/mL The “fluid density” can be measured conventionally by calibrating a scale to zero with the container thereon and then obtaining the mass of the fluid, e.g., liquid, in grams. The volume of the measure mass can then be determined using a graduated cylinder. The density is then calculated by dividing the mass by the volume to get the fluid density (g/mL).
The vertically layered fluid column 100 also includes a third fluid 120 in this example. However, in this instance, the density of the third fluid is less dense than the second fluid 170. Thus, in a more standard sized column, the third fluid may otherwise migrate up into or through the second fluid, destroying the interface between the second and third fluids. However, in the example shown, this is not the case. The third fluid is constrained by the cross-sectional size of the vessel that contains the vertically layered fluid column (along the plane where the second fluid interfaces with the third fluid). Thus, the surface tension of the third fluid combined with the size constraint of the column at this interface in combination provide capillary force-supported interface 125, which promotes the second fluid and the third fluid remaining separated from one another. More specifically, the capillary force-supported interface can be contained within a fluidic channel having a cross-sectional dimension that is aligned with the capillary force-supported interface, and can be, for example, less than 2 mm. This dimension can be a diameter dimension for circular channels, or for non-circular geometries, this dimension can be the average cross-sectional dimension or the distance between opposing parallel surfaces of the channel, for instance. If the channel at this location is conical, the distance along the capillary force-supported interface can be used.
The first fluid 160, the second fluid 170, and the third fluid 120 can be any of a number of combinations of fluids, such as gas fluid, e.g., air, aqueous fluid, non-polar fluid, master mix fluid, reagent fluid, surfacing binding fluid, washing fluid, elution fluid, lysis fluid, etc. These or other fluids may be selected for use, and furthermore, these or other fluids may likewise be selected for use either above the first fluid or beneath the third fluid, as shown in
Referring now to
In further detail regarding the multi-fluid density gradient portion 101 of the vertically layered fluid column 100, there can be any of a number of fluids in this portion of the column, e.g., two fluids, three fluids, four fluids, etc., vertically arranged. Thus, a “multi-fluid density gradient portion” as used herein, can refer to a multi-layered fluid arranged with density gradient interfaces extending horizontally there between. The fluids may or may not be positioned 90 degrees from horizontal relative to one another, e.g., they may or may not be stacked or layered directly on top of one another but may be in a vessel angled at less than 90 degrees from horizontal, but the interface between the fluids are essentially horizontal. Thus, the term “vertically layered” refers to fluids that are on top of one another relative to a force such as gravity or centripetal force in a centrifuge with a horizontal interface extending there between, even if they are not fully directly on top of one another. A multi-fluid density gradient portion of the vertically layered fluid column does not include fluid layers where an additional substance may be used to separate one fluid layer from another. Fluid layers of the multi-fluid density gradient portion can be phase separated from one another based on fluidic properties of the various fluids, including density of the respective fluids along the column. The greater or higher the density of a fluid, relative to other fluids in the column, the closer to the bottom of the column the fluid will be located as defined or established by gravity. For example, the first fluid layer can have a first density and can form a first fluid layer of the multi-fluid density gradient portion. The second fluid layer can have a second density that can be greater than a density of the first fluid layer and can form a second fluid layer of the multi-fluid density gradient portion beneath the first fluid layer. An additional fluid layer(s), e.g., third, fourth, etc., can have a third, fourth, etc., densities that can be greater than a density of the previous fluid layer and can form a third, fourth, etc., fluid layer of the multi-fluid density gradient portion beneath the second fluid layer. As a note, this is not the case for the “capillary force-supported interface.” In that instance, the surface tension of the fluid relative to the size and material of the vessel provides the ability to put less dense fluids beneath fluids of greater density, e.g., below the multi-fluid density gradient portion (see
In further detail regarding the vessels that can be used to support the vertically layered fluid column used, they can be configured as shown in
In further reference to the multi-fluid density gradient portion of the vertically layered fluid column, in some examples, a density of a fluid in a fluid layer can be altered using a densifier. Example densifiers can include sucrose, polysaccharides such as FICOLL™ (commercially available from Millipore Sigma (USA)), C19H26I3N3O9 such as NYCODENZ® (commercially available from Progen Biotechnik GmbH (Germany)) or HISTODENZ™, iodixanols such as OPTIPREP™ (both commercially available from Millipore Sigma (USA)), or combinations thereof. In one example, a density difference of the first fluid layer relative to the second fluid layer can be from about 50 mg/mL to about 3 g/mL. In yet other examples, a density difference from the first fluid layer relative to the second fluid layer can be from about 50 mg/mL to about 500 mg/mL or from about 250 mg/mL to about 1 g/mL. In further detail, example additives that can be included in the first fluid layer, or in other fluid layers, depending on the design of the multi-fluid gradient column may include sucrose, C1-C4 alcohol, e.g., isopropyl alcohol, ethanol, etc., which can be included to adjust density, and/or to provide a function with respect to biological components or materials to pass through the column.
A quantity of fluid layers in the multi-fluid density gradient portion and/or the capillary force-supported gradient portion is not particularly limited. In one example, the multi-fluid density gradient portion can further include a fourth fluid layer having a fourth fluid density that can be greater than a third fluid with a third fluid density and can be positioned beneath the third fluid layer. The fourth fluid layer can be phase separated from the third fluid layer along a third fluid layer interface where the third fluid layer can be in contact as a layer relative to the fourth fluid layer. In further examples, the assembly can further include a fifth, sixth, or seventh fluid layer that can be phase separated from the other fluids in the column based on a density of the fifth, sixth, or seventh fluid with respect to the other fluids in the column.
In accordance with examples of the present disclosure, a few example biological component processing systems 200 are shown in
Referring initially to
The density difference between fluid 160 and fluid 170 can be sufficient so that the fluids remain phase separated at a first density-differential interface 115A, and the density difference between the fluid 170 and fluid 180 can be sufficient so that the fluids remain phase separated at a second density differential interface 1156, even though the fluids are in direct contact or in direct fluid communication layered on top of one another, e.g., the fluids are separated by their densities, not by a membrane or other artificial structure therebetween. Example density differences of any two fluids along the multi-fluid density gradient portion can be from 50 mg/mL to 3 g/mL, from 100 mg/mL to 3 g/mL, from 500 mg/mL to 3 g/mL or from 1 g/mL to 3 g/mL.
The vertically layered fluid column 100 also includes fluid 120 in this example. However, in this instance, the density of fluid 120 is less than the fluid 170, even though it is positioned immediately there beneath. Thus, in a more standard sized column, fluid 120 may otherwise migrate up into or through the second fluid 170, destroying the interface between the fluids. However, in the example shown, this is not the case, as fluid 120 is constrained by the cross-sectional size of the column structure (along the plane where the second fluid interfaces with the third fluid). Thus, the surface tension of fluid 120 combined with the size constraint of the column at this interface provides a (first) capillary force-supported interface 125A, which promotes fluid 170 and fluid 120 remaining separated from one another. More specifically, the capillary force-supported interface can be contained within a fluidic channel having a cross-sectional dimension that is aligned with the capillary force-supported interface, and can be, for example, less than 2 mm, from 1 μm to 1 mm, from 1 mm to 1.75 mm, from 0.75 μm to 100 μm, or from 1 μm to 50 μm. This dimension can be a diameter dimension, or for non-circular geometries, this dimension can be the average cross-sectional dimension. Again, fluid 160, fluid 170, fluid 120, or any of the other fluids can be any of a number of combinations of fluids, such as gas fluid, e.g., air, aqueous fluid, non-polar fluid, polar, non-polar, miscible, immiscible, etc. The fluids can be, for example a master mix fluid, reagent fluid, surfacing binding fluid, washing fluid, elution fluid, lysis fluid, etc. The fluids can likewise be pure, solutions, mixtures, suspensions, emulsions, and/or in other forms. They may or may not undergo chemical reactions within the vessel at any stage of the process, depending on the application.
In further detail, in the example shown in
In further detail, individual fluids in the various layers can provide different functions, regardless of the orientation, e.g.,
In some examples, an individual fluid in one or multiple layers can provide sequential processing of a biological component from a biological sample. For example, individual fluids can carry out individual functions, and in many cases, the functions can be coordinated to achieve a specific result. Biological samples that may be added can include whole blood, platelets, cells, lysed cells, cellular components, tissue, nucleic acids, e.g., DNA, RNA, primers, oligos, etc., or poly-bases, peptides, or the like. More specifically, for example, in considering biological components of interest and second components found in a cell, sequential fluid from top to bottom of a multi-fluid density gradient portion can act on the cell to lyse the cell in one of the fluids, and bind a target biological component from the lysed cell to particulate substrate, e.g., magnetizing particles, in a second fluid (or lysing and binding can alternatively be done in a single fluid). Additional fluid may be used to wash the particulate substrate with the biological component bound thereto in another fluid, e.g., washing the second fluid from particulate substrate in the next fluid, and/or eluting (or separating) the biological component from the particulate substrate in yet another lower layer. The surface binding and cell lysis can occur, for example, with a lysate buffer in a sucrose and water solution, e.g., the lysate (lysis) buffer can be densified with sucrose. Washing can occur in a sucrose in water solution, for example. In other examples, one or more of the fluids can be present as a fluid (layer(s)) along the multi-fluid density gradient portion in the form of a master mix fluid for nucleic acid processing. Other combinations of fluids (first, second, third, etc.) may include a surfacing binding fluid, a washing fluid, and an elution fluid; or may include a lysis fluid, a washing fluid, a surface binding fluid, a second washing fluid, an elution fluid, and a reagent fluid. Regardless of the various functions of the various fluids with sequentially increasing densities arranged from top to bottom, at the individual fluids, the particulate substrate can independently interact, e.g., become modified, with fluids as layers in order to sequentially process the particulate substrate with surface active groups and/or biological components associated therewith or associated with one or more of the fluids, for example.
A vertical height of the various layers of fluids in the multi-fluid density gradient portion can vary. Adjusting a vertical height of a fluid layer can affect a residence time of the magnetizing particles, e.g., paramagnetic microparticles, in that fluid layer. The taller the fluid layer, the longer the residence time of the magnetizing particles in the fluid layer. Notably, the speed at which the magnets move may also be adjustable. In some examples, all of the fluid layers in the multi-fluid density gradient portion can be the same vertical height. In other examples, a vertical height of individual fluid layers in a multi-fluid density gradient portion can vary from one fluid layer to the next. In one example, a vertical height of the various layers along the multi-fluid density gradient portion can individually be from about 10 μm to about 50 mm. In another example, a vertical height of the fluid layers along the multi-fluid density gradient portion can individually be from about 10 μm to about 30 mm, from about 25 μm to about 1 mm, from about 200 μm to about 800 μm, or from about 1 mm to about 50 mm.
The biological component processing systems 200 shown in
In further detail regarding the “particulate substrate,” these materials can be in the form of particles, such as microparticles. These particles can be denser than the various fluids along the vertically layered fluid column so that they move in a negative z-axis direction, e.g., glass, silica, etc., or can be less dense or buoyant so they can move upward through the vertically layered fluid column in a positive z-axis direction. The particulate substrate can also be particles that are “magnetizing” meaning that when a magnetic field is applied, they can respond and move with the magnetic field when a static magnetic field is applied and/or can move as the magnetic field is modified dynamically. Movement of the magnetizing particles can be a positive or negative z-axis direction. The surface morphology of the particulate substrate may be smooth or rough, and dimensions may vary from sub-micron, e.g., from 100 nm or 500 nm, to about 20 μm. The particulate substrate may, for example, include surfaces that bind to nucleic acids either reversibly or permanently (relative to downstream processing fluids selected for use). The particulate substrate may be in the form of glass or silica particles May bind NA reversibly or permanently, based on downstream process requirements.
The particulate substrate can include surfaces that are bound to or otherwise associated with a biological component or can be formulated to become bound to or otherwise associated with a biological component in situ.
The term “associated” refers to any type of attach or adherence of a biological component with a surface of the particulate substrate. This can include covalent bonding, electrostatic or ionic attraction, surface adsorption, hydrogen bonding, and/or other adherence or linkage suitable for moving biological component together with the particulate substrate. In accordance with this, in some examples, the system can include a biological sample and the particulate substrate (magnetizing particles or otherwise) can be surface activated to preferentially bind with a biological component relative to secondary components in a biological fluid sample. Thus, the biological component may preferentially bind to the surface compared to secondary components such as enzymes, cellular debris, lysing agents, buffers, or a combination thereof. The particulate substrate can be loaded in any of the fluid layers and moved vertically in either direction (up or down). As shown in
In further detail regarding the particulate substrate, as mentioned, the particulate substrate can be particles with a density suitable for gravity settling or centrifugation separation or movement along the column. In some examples, the particulate substrate can be in the form of magnetizing particles. Magnetizing particles can be in the form of paramagnetic microparticles, superparamagnetic microparticles, diamagnetic microparticles, or a combination thereof, for example. Whether using magnetizing particles or otherwise, the particulate substrate can be surface-activated to bind with a biological component or can be bound to the biological component. The particulate substrate can be surface activated, for example, with surface groups that are interactive with a biological component of a biological sample or can include a covalently attached ligand attached to a surface of the particulate substrate to likewise bind with a biological component of a biological sample. In some examples, the ligand can include proteins, antibodies, antigens, nucleic acid primers, amine groups, carboxyl groups, epoxy groups, tosyl groups, sulphydryl groups, silane groups, poly dT oligomers, target-specific oligomers, streptavidin, or the like.
Regarding combinations of ligands, there can be multiple types of ligands on a common particulate substrate, a mixture of particulate substrate with different ligands on multiple portions of the particulate substrate (with the same or different particulate substrate). The ligand can be selected to correspond with and bind with the biological component and can vary based on the type of biological component being isolated from the biological sample. For example, the ligand can include a nucleic acid primer when isolating a biological component that includes a nucleic acid sequence. In another example, the ligand can include an antibody when isolating a biological component that includes antigen. By way of example, commercially available examples of magnetizing particles with surface-activated groups include those sold under the trade name DYNABEADS®, available from ThermoFischer Scientific (USA).
In some examples, the particulate substrate can have an average particle size that can be from about 0.1 μm to about 70 μm. The term “average particle size” morphology of the individual particle. A shape of the particulate substrate can be spherical, irregular spherical, rounded, semi-rounded, discoidal, angular, sub-angular, cubic, cylindrical, or any combination thereof. In one example, the particles can include spherical particles, irregular spherical particles, or rounded particles. The shape of the particulate substrate can be spherical and uniform, which can be defined herein as spherical or near-spherical, e.g., having a sphericity of >0.84. Thus, any individual particles having a sphericity of <0.84 are considered non-spherical (irregularly shaped). The particle size of the substantially spherical particle may be provided by its diameter, and the particle size of a non-spherical particle may be provided by its average diameter (e.g., the average of multiple dimensions across the particle) or by an effective diameter, e.g., the diameter of a sphere with the same mass and density as the non-spherical particle. In further examples, the average particle size of the particulate substrate can be from about 1 μm to about 50 μm, from about 5 μm to about 25 μm, from about 0.1 μm to about 30 μm, from about 40 μm to about 60 μm, or from about 25 μm to about 50 μm.
In an example, the particulate substrate can be unbound to a biological component when added directly to one of the fluid (layers) of a multi-fluid density gradient portion and/or the capillary force gradient portion. Binding between the particulate substrate and the biological component of the biological sample can occur in the multi-fluid density gradient portion and/or the capillary force gradient portion. In yet another example, the particulate substrate and a biological sample including a biological component can be combined in a loading fluid before being added to a multi-fluid density gradient portion. In this example, binding of the particulate substrate to the biological component of the biological sample can occur in the multi-fluid density gradient portion.
With more specific detail regarding the magnetizing particles, the term “magnetizing particles” is defined herein to include microparticles that may not be magnetic in nature unless and until a magnetic field is introduced at a strength and proximity to cause them to become magnetic. Their magnetic strength can be dependent on the magnetic field applied and may get stronger as the magnetic flied is increased, or the magnetizing particles get closer to the magnetic source that is applying the magnetic field. In more specific detail, “paramagnetic microparticles” have these properties, in that they have the ability to increase in magnetism when a magnetic field is present; however, paramagnetic microparticles are not particularly magnetic when a magnetic field is not present. In some examples, the paramagnetic microparticles can exhibit no residual magnetism once the magnetic field is removed. A strength of magnetism of the paramagnetic microparticles can depend on the strength of the magnetic field, the distance between a source of the magnetic field and the paramagnetic microparticles, and a size of the paramagnetic microparticles. As a strength of the magnetic field increases and/or a size of the paramagnetic microparticles increases, the strength of the magnetism of the paramagnetic microparticles increases. As a distance between a source of the magnetic field and the paramagnetic microparticles increases the strength of the magnetism of the paramagnetic microparticles decreases. “Superparamagnetic microparticles” can act similar to paramagnetic microparticles; however, they can exhibit magnetic susceptibility to a greater extent than paramagnetic microparticles in that the time it takes to become magnetized appears to be near zero seconds. “Diamagnetic microparticles,” on the other hand, can display magnetism due to a change in the orbital motion of electrons in the presence of a magnetic field.
The vertically layered fluid column can be part of a system that includes magnetizing particles, which can be, for example, paramagnetic microparticles, superparamagnetic microparticles, diamagnetic microparticles, or a combination thereof. Paramagnetic microparticles can have the ability to increase in magnetism when a magnetic field is present; however, paramagnetic microparticles are not magnetic when a magnetic field is not present. In some examples, the paramagnetic microparticles can exhibit no residual magnetism once the magnetic field is removed. The strength of magnetism of the paramagnetic microparticles can depend on the strength of the magnetic field, the distance between a source of the magnetic field and the paramagnetic microparticles, and the size of the paramagnetic microparticles. As the strength of the magnetic field increases and/or the size of the paramagnetic microparticles increases, the strength of the magnetism of the paramagnetic microparticles will be larger. As the distance between the source of the magnetic field and the paramagnetic microparticles increases, the strength of the magnetism of the paramagnetic microparticles decreases.
Superparamagnetic microparticles can act similar to paramagnetic microparticles; however, they can exhibit magnetic susceptibility more quickly than paramagnetic microparticles in that the magnetization time appears to be near zero seconds. Diamagnetic microparticles can display magnetism due to a change in the orbital motion of electrons in the presence of a magnetic field.
Referring now to
In further detail regarding
Thus, in one example, as shown, the biological component processing system 200 of
The vertically layered fluid columns and/or biological component processing systems described herein can be used or can further include a magnet, such as in the form of electrically induced magnet field generating element(s), permanent magnet(s), or a combination thereof. An electrically induced magnetic element can be, for example, turned on and off by introducing electrical current/voltage to the element and generating a magnetic field. Alternatively, the magnet can be a permanent magnet that is placed in proximity to the multi-fluid density gradient portion to effect and/or affect the movement of the magnetizing particles. In either instance, the magnet can be moved physically to cause movement of magnetizing particles along the vertically layered fluid column. Thus, magnet can be permanently placed within this proximity, or can be movable along the column, or movable in position and/or out of position to effect movement of the magnetizing particles. The magnetizing particles can be magnetized by the magnetic field generated by the magnet or electrically induced magnet. In addition, the magnet can create a force capable of pulling the magnetizing particles through the multi-fluid density gradient portion. When the magnet is turned off or is not in appropriate proximity, the magnetizing particles can reside in a fluid layer until gravity pulls the magnetizing particles through fluid layers of the multi-fluid density gradient portion, or they may remain suspended in the fluid layer in which they may reside until the magnetic field is applied thereto. The rate at which gravity pulls the magnetizing particles through fluid layers (or leaves the magnetizing particles within a fluid layer) can be based on a mass of the magnetizing particles in combination with a surface tension between fluid layers and/or density of the magnetizing particles and density of the fluid layers. The magnet can cause the magnetizing particles to move from one fluid layer to another or increase a rate at which the magnetizing particles pass from one fluid layer into another.
In an example, the magnet 190 can be positioned above (
A strength of the magnetic field and the location of the magnet in relation to the magnetizing particles can affect a rate at which the magnetizing particles move downwardly through the multi-fluid density gradient portion. The further away the magnet and the lower the strength of the magnetic field, the slower the magnetizing particles will pass through the multi-fluid density gradient portion. In an example, a maximum distance between the magnet and a nearest location where one or more of the fluids resides along the multi-fluid density gradient portion can be about 50 mm maximum distance, about 40 mm maximum distance, about 30 mm maximum distance, about 20 mm maximum distance, or about 10 mm maximum distance. The minimum distance, on the other hand, may be from about 0.1 mm minimum distance, from about 1 mm minimum distance, or from about 5 mm minimum distance. In one example, the minimum distance between the magnet and the multi-fluid density gradient portion may be about the thickness of the vessel that contains the multi-fluid density gradient portion. Thus, distance ranges between the magnet and the multi-fluid density gradient portion can be from about 0.1 mm to about 50 mm, from about 1 mm to about 50 mm, from about 1 about mm to about 40 mm, from about 1 mm to about 30 mm, from about 1 mm to about 20 mm, from about 1 mm to about 10 mm, from about 5 mm to about 50 mm, or from about 5 mm to about 30 mm. In another example, a maximum distance between the magnet and a nearest location where one of the fluids resides along the multi-fluid density gradient portion can be about 30 mm.
Methods of Processing Biological Components from Biological Samples
A flow diagram of a method 300 of processing a biological component from a biological sample is shown in
In one example, the biological component can be associated with a particulate substrate that is passed through the density-differential interface and the capillary force-supported interface via a force selected from gravity, a centrifugal force, a magnetic field, buoyance, or a combination thereof. In another example, the biological component can be associated with a particulate substrate that is passed through the density-differential interface and the capillary force-supported interface via a magnetic field. The term “processing” can refer to concentrating, separating, diluting, amplifying, washing, lysing, decontaminating, fluid exclusion, eluting, or carrying out any other process where the biological component is modified in some way within the vertically layered fluid column.
In one example, the biological component can be bound to or otherwise associated with a particulate substrate that is passed through the density-differential interface and the capillary force-supported interface via a force selected from gravity, a centrifugal force, a magnetic field, buoyancy, or a combination thereof. In some other examples, the biological sample including the biological component can be combined with the magnetizing particles in a loading solution prior to loading the biological sample including the biological component and the magnetizing particles into the multi-fluid density gradient portion. For example, the magnetizing particles and the biological sample can be admixed in a loading fluid. The biological sample and the magnetizing particles can be permitted to incubate or otherwise become prepared for loading on top of or into the multi-fluid density gradient portion. The magnetizing particles can bind with the biological component in the loading fluid and can then be added to the multi-fluid density gradient portion for the fluid layers to act upon the magnetizing particles. In one example, the loading fluid can become the uppermost fluid layer when loading from the top or can become the lowermost fluid layer when loading from the bottom, for example. Other fluid layers beneath or above the loading layer can be included through which the particulate substrate is passed in part or in full.
The fluid used for loading the column (or the first fluid, or even the second fluid, or other fluid layer) can include secondary components selected from enzymes, cellular debris, lysing agents, buffers, or a combination thereof. The magnetizing particles can be bound to the biological component in a loading fluid or in a subsequent fluid along the multi-fluid density gradient portion. In the case of a loading fluid, magnetizing particles including the biological component bound thereto can then be introduced as a separate fluid layer for the microparticles to be drawn into other fluid layers that can act on the microfluidic particles to further interact with the surface thereof along the multi-fluid density gradient portion.
In accordance with the method, the particulate substrate can be passed through multiple density-differential interfaces and/or multiple capillary force-supported interfaces, depending on the arrangement. The particulate substrate can be passed through any or all of these interfaces from fluid to fluid in an upward or downward z-axis direction, though movement along the x- and y-axes can also occur during the movement of the particles upward or downward. It is noted once again that the vertically layered fluid column can be arranged with layered fluids in an orientation of about 90 degrees from horizontal, or the column can be at any angle suitable for upward or downward movement of the particulate substrate through horizontal fluid interfaces.
In one example, the method can further include selectively withdrawing, e.g., pipetting, the biological component out of the third fluid layer, such as through an ingress/egress opening(s) from the top, the bottom, or through a sidewall, for example. The biological component may still be associated with a surface of the magnetizing particles or may be separated from the magnetizing particles. In another example, this method alternatively may include selectively withdrawing, e.g., pipetting, from one of the fluids (one of the layers), the second fluid layer, and/or the third fluid layer out of the multi-fluid density gradient portion and leaving the magnetizing particles with the biological component bound thereto in a vessel of the multi-fluid density gradient portion to either be further treated or removed after the extraction of one of the fluids, the second fluid layer, and the third fluid layer therefrom. In some examples, the biological sample can include a cell with the biological component trapped within the cell (prior to lysis), a virus, or a biological component with extra-cellular vesicles. Lysing the cell can release the biological component therefrom and can permit isolation of the biological component. In this example, one of the fluids (or a loading fluid) can include a lysing agent for the cell. The method can further include lysing the cell in situ within one of the fluids or the loading fluid so that the biological component can be liberated from the cell and can bind with the magnetizing particles in one of the fluids (or fluid layers) or the loading fluid.
As mentioned, fluid 160, fluid 170, fluid 120, or any of the other fluids can be any of a number of combinations of fluids, such as gas fluid, e.g., air, aqueous fluid, non-polar fluid, master mix fluid, reagent fluid, surfacing binding fluid, washing fluid, elution fluid, lysis fluid, etc. However, in this example, various processes are shown in sequence to provide an example of the use of the present technology. Notably, magnets are not shown, but if magnetizing particles are used as the particulate substrate, then a magnet or series of magnets can be used to draw the particles in a controlled manner in the negative z-direction.
In accordance with the general processing stream shown in
The nucleic acid from the cell can become bound to a surface of the particulate substrate 210, which can be a magnetizing particle (M), for example. Next, shown at (B), as the particles are drawn through interface 115B, cellular debris and unbound nucleic acid can be exchanged for a wash buffer at fluid 160. A second wash can occur at fluid 170, shown at (C), as bead-bound nucleic acid is further cleared of contaminants, e.g., lysed cellular debris and/or other contaminants or other material not of interest that may be present. Example wash buffers for use at fluids 180 and/or 160 can be:
In further detail regarding the specific example shown in
Regarding the oil layer in the capillary force gradient portion of the column, shown at (D), specific oils that can be used include:
Once the particulate substrate passes through the oil, a gas layer, which in this instance can be air (or an air gap), can be used to clear the contaminants further and can provide a mechanism to provide little to no contact between the fluids above and below the air gap. The biological component being separated (or further processed) has now have been loaded on the particulate substrate after cell lysis, washed in two different wash buffer layers, further contaminant-cleared by the oil, and passed through the air gap, providing reduced likelihood of concentration of contact between wash and/or lysis buffer and the next fluid beneath the air gap, e.g., elution buffer, a master mix fluid for nucleic acid processing, or the like. To separate the biological component, e.g., nucleic acid, from the particulate substrate, an elution buffer can be used. Example elution buffers suitable for use may include one or more of:
It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint. The degree of flexibility of this term can be dictated by the particular variable and determined based on experience and the associated description herein.
As used herein, the term “interact” or “interaction” as it relates to a surface of the particulate substrates, such as the magnetizing particles, and indicates that a chemical, physical, or electrical interaction occurs where a particulate substrate surface property is modified in some manner that is different than may have been present prior to entering the fluid layer, but does not include modification of magnetic properties magnetizing particles as they are influenced by the magnetic field introduced by the magnet. For example, a fluid layer can include a lysis buffer to lyse cells, and cellular components can become bound to or otherwise associated with a surface of the magnetizing particles. Lysing cells in a fluid can modify the fluid sample and thus modify or interact with a surface of magnetizing particles, e.g., the cellular component binds or becomes otherwise associated with a surface of the magnetizing particles. In one example, the association between the biological component and the magnetizing particles (or other particulate substrate) can alternative include surface adsorption, electrostatic attraction, or by some other attraction between biological component and the surface of the particulate substrate. In yet other examples, a fluid layer that would be considered to interact with the magnetizing particles could be a wash fluid layer to trap contaminates from a sample fluid and/or remove contaminates from an exterior surface of the magnetizing particles, a surfactant fluid layer to coat the magnetizing particles, a dye fluid layer to introduce visible or other markers to the fluid or surface, an elution fluid layer to remove the biological component from the magnetizing particles following extraction from the biological sample, a labeling fluid layer for binding labels to the biological component such as a fluorescent label (either attached to the magnetizing particles or unbound thereto), a reagent fluid layer to prep a biological component for further analysis such as a master mix fluid layer to prep a biological component for PCR, and so on.
As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though individual members of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on presentation in a common group without indications to the contrary.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. A range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include individual numerical values or sub-ranges encompassed within that range as if numerical values and sub-ranges are explicitly recited. As an illustration, a numerical range of “about 1 wt % to about 5 wt %” should be interpreted to include not only the explicitly recited values of about 1 wt % to about 5 wt %, but also to include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3.5, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc. This same principle applies to ranges reciting one numerical value. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
While the present technology has been described with reference to certain examples, various modifications, changes, omissions, and substitutions can be made without departing from the spirit of the disclosure. The disclosure is limited only by the scope of the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/032187 | 5/8/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/058429 | Oct 2019 | US |
| Child | 17642265 | US | |
| Parent | PCT/US2019/058427 | Oct 2019 | US |
| Child | PCT/US2019/058429 | US |
