WO2022125068A1

WO2022125068A1 - Isolating biological components

Info

Publication number: WO2022125068A1
Application number: PCT/US2020/063725
Authority: WO
Inventors: Si-Lam J. Choy; John Michael LAHMANN; David Daniel Welter
Original assignee: Hp Health Solutions Inc.
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2022-06-16

Abstract

A method of isolating a biological component from a biological sample can include dispensing a sample fluid including magnetizing particles and a wash buffer into interconnected volumes including a bulk fluid volume fluidically connected in series with a capillary volume to form a density gradient column, the wash buffer having a greater density and positioned beneath the sample fluid and the density gradient column including magnetizing particles. The method can include magnetically moving the magnetizing particles from the sample fluid into the wash buffer at a location residing within the capillary volume, partitioning a downstream portion of the wash buffer containing the magnetizing particles in the capillary volume from a balance of the wash buffer thereabove to form a partitioned sample fluid that includes the magnetizing particles, and dispensing a fluid reagent or a dried reagent into the capillary volume to interact with the partitioned sample fluid.

Description

ISOLATING BIOLOGICAL COMPONENTS

BACKGROUND

[0001] 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 the component of interest. As the quantity of available assays increases, so does the demand for the ability to isolate components of interest from sample fluids.

BRIEF DESCRIPTION OF THE DRAWINGS

[0002] FIG. 1 is a flow diagram of an example method of isolating a biological component from a biological sample in accordance with the present disclosure;

[0003] FIGS. 2A-2J graphically illustrate example methods of isolating a biological component from a biological sample in accordance with the present disclosure; and

[0004] FIG. 3 graphically illustrates an example system for isolating a biological component from a biological sample in accordance with the present disclosure.

DETAILED DESCRIPTION

[0005] Biological components 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 obtained for analysis from a living or deceased organism. Isolating a biological component from other components of the biological sample can permit subsequent analysis of the isolated biological component without interference from the other components in the biological sample and can increase an accuracy of the subsequent analysis of the isolated biological component. In addition, isolating the biological component from other components in the biological sample can permit analysis of the biological component that would not be possible if the biological component was not readily accessible within the biological sample. Many 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 result in less than maximum yields of the isolated biological component.

[0006] The present disclosure describes methods for isolating a biological component from a biological sample, systems for isolating a biological component from a biological sample, and devices that can be used in a more specific process of preparing samples for a PCR (polymerase chain reaction) assay. PCR assays are processes that can rapidly copy millions to billions of copies of a very small DNA or RNA sample. PCR can be used for many different application, included sequencing genes, diagnosing viruses, identifying cancers, and others. In the PCR process, a small sample of DNA or RNA is combined with reactants that can form copies of the DNA or RNA. Because the volumes of samples fluid and reactant involved in this process are very small, it can be beneficial to use small fluidic devices and systems such as those described herein.

[0007] In accordance with example methods of the present disclosure, isolating a biological component from a biological sample includes dispensing a sample fluid including magnetizing particles and a wash buffer into interconnected volumes including a bulk fluid volume fluidically connected in series with a capillary volume to form a density gradient column. The wash buffer has a greater density and is positioned beneath the sample fluid within the interconnected volumes. The density gradient column in this example includes magnetizing particles. In some examples, the biological component of interest, coming from the biological sample, may be a nucleic acid (such as DNA or RNA). The density gradient column also includes, for example, magnetizing particles (initially present in the sample fluid). The method also includes also magnetically moving the magnetizing particles from the sample fluid into the wash buffer at a location residing within the capillary volume. As a note, the magnetizing particles may be linked or associated with a biological component of interest, e.g., a nucleic acids, and thus, the sample fluid may be a biological sample fluid. In further detail, the method includes partitioning a downstream portion of the wash buffer containing the magnetizing particles in the capillary volume from a balance of the wash buffer thereabove to form a partitioned sample fluid that includes the magnetizing particles, and dispensing a fluid reagent or a dried reagent into the capillary volume to interact with the partitioned sample fluid.

[0008] In one example, the method can further include preparing the magnetizing particles by selectively binding a biological component to surface-activated magnetizing particles, where the surface-activated magnetizing particles include interactive surface groups or a ligand thereon complimentary to the biological component. In some examples the ligand is a nucleic acid strand that is complementary to the target nucleic acid which is present in the biological sample. In other examples, the method can further include lysing a biological sample to release the biological component that was contained therein, where the lysing includes heating the biological sample, chemically lysing the biological sample, or a combination thereof. In yet another example, the method can further include admixing the biological component released from the biological sample with the magnetizing particles to bind the biological component to interactive surface groups or ligands on the magnetizing particles. In a further example, the partitioning can include dispensing a gas into the fluid capillary to form a separation gas bubble between the portion of the wash buffer above the partitioned sample fluid and the partitioned sample fluid. In a further example, the partitioning can include dispensing a non-newtonian plugging fluid into the fluid capillary to form a fluid plug. In one example, the method can further include dispensing the partitioned sample fluid combined with the fluid reagent or a reconstituted dried reagent from the capillary volume through an output channel. The partitioned sample fluid may include the biological component bound to magnetizing particles during the dispensing from the capillary volume, in an example. In another example, the magnetizing particles can have the biological component bound thereto and the method can further include heating the magnetizing particles and the fluid reagent to separate a biological component from magnetizing particles in the capillary volume. In yet another example, the method can further include applying a magnetic field to the capillaryvolume to trap the magnetizing particles therein, and dispensing the separated biological component from the capillary volume through an output channel. In a further example, the partitioning of the magnetizing particles and the dispensing of the fluid reagent includes, reconstituting dried reagent stored within a dry reagent reservoir. In one example, the partitioning of the magnetizing particles and the dispensing of the fluid reagent includes forcing a gas and reconstituted fluid reagent from the dry reagent reservoir into the fluid capillary. In another example, the dispensing of the fluid reagent into the capillary volume can occur downstream of where the magnetizing particles are partitioned in the partitioned sample fluid. In yet another example, the magnetizing particles can be present in the sample fluid when dispensing the sample fluid. In another example, dispensing the sample fluid and the wash buffer can include dispensing the wash buffer into the capillary volume and upward into a bulk fluid volume positioned thereabove, and dispensing the sample fluid over the wash buffer to form the density gradient column.

[0009] A system for isolating a biological component from a biological sample includes, in one example, magnetizing particles that are surface-activated to bind with a biological component, or which are bound to the biological component; and interconnected volumes to receive or which contain a density gradient column including a sample fluid positioned or positionable above a wash buffer. The wash buffer has a greater density than the sample fluid, and the interconnected volumes include a bulk fluid volume positioned in series with a capillary volume. The system also includes a dry reagent reservoir with a dried reagent or a fluid reagent reservoir including a fluid reagent. The dry reagent reservoir or the fluid reagent reservoir is positioned outside and f lu idically connected to the capillary volume. A magnetic field generator is also included in this example to draw the magnetizing particles along the density gradient column from the sample fluid into the wash buffer contained within the capillary volume. In one example, the dried reagent can include nucleic acid primers, deoxynucleosides, triphosphates, reverse transcriptase, secondary antibodies, polymerases, enzymes, polymerases, probes, magnesium salt, bovine serum albumin (BSA), beads, or combinations thereof. In one example, the system can further include a wash buffer reservoir, an air reservoir, a non-newtonian plugging fluid reservoir, a reconstitution buffer reservoir, or a combination thereof located outside of the density gradient column and in fluidic connection with the density gradient column. In yet another example, the dry reagent reservoir can include a reconstitution buffer injection opening, or the system can further include a reconstitution buffer reservoir positioned upstream of the dry reagent reservoir and fluidically connected to the dry reagent reservoir. In one example, a reconstituted fluid reagent can include the dried reagent and a reconstitution buffer can have a density less than the wash buffer. In an example, the system can further include a cap coupled to an end of the capillary volume at a biological sample output.

[0010] It is noted that when discussing a method of isolating a biological component from a biological sample or the system for isolating a biological component from a biological sample herein, 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 density gradient column, such disclosure is relevant to and directly supported in the context of the method of isolating a biological component from a biological sample, or the system for isolating a biological component.

[0011] 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.

[0012] The present disclosure includes several figures illustrating specific examples of the technologies described herein. These figures show fluidic devices and fluid processing systems that include a variety of components arranged is specific ways depending on the purpose and function of the particular exampies depicted. Although the figures illustrate examples that implement the technologies described herein, these examples also include many features that are optional, which may be changed or removed depending on the particular example. Accordingly, it is understood that the technologies described herein are not limited by the examples shown in the figures.

Methods of Isolating a Biological Components in Biological Samples

[0013] A flow diagram 100 of a method of isolating a biological component from a biological sample is shown in FIG. 1. The method can include dispensing 110 a sample fluid including magnetizing particles and a wash buffer into interconnected volumes including a bulk fluid volume fluidically connected in series with a capillary volume to form a density gradient column. The wash buffer has a greater density and is positioned beneath the sample fluid within the interconnected volumes. The density gradient column in this example includes magnetizing particles. In some examples, the biological component of interest, coming from the biological sample, may be a nucleic acid (such as DNA or RNA). The density gradient column can also include, for example, magnetizing particles (initially present in the sample fluid). The method further includes magnetically- moving 120 the magnetizing particles from the sample fluid into the wash buffer at a location residing within the capillary volume. As a note, the magnetizing particles may be linked or associated with a biological component of interest, e.g., a nucleic acids, and thus, the sample fluid may be a biological sample fluid. In further detail, the method includes partitioning 130 a downstream portion of the wash buffer containing the magnetizing particles in the capillary volume from a balance of the wash buffer thereabove to form a partitioned sample fluid that includes the magnetizing particles, and dispensing 140 a fluid reagent or a dried reagent into the capillary volume to interact with the partitioned sample fluid. The sample fluid can include DNA, RNA, proteins, viruses, antibodies, or a variety of other biological materials. In one particular example, the method can be used to detect a virus and the biological sample can include RNA or DNA extracted from the virus. The RNA or DNA can be extracted by lysing viruses, which can result in a solution containing the viral RNA or viral DNA in addition to fragments of lysed viruses and other materials. The wash buffer can be a liquid that can be used to wash a biological sample.

[0014] FIGS. 2A-2J schematically illustrate various example methods of isolating a biological component from a biological sample. Though some detail is shown in these FIGS., not every step is necessarily relevant to all possible methods. Various combinations of steps, sequences, or other variables can be practiced in accordance with the example provided in FIGS. 2A-2J. Furthermore, in order to provide additional clarity, the numerical references in FIGS. 2A-2J should be viewed collectively, even if not specifically described or shown for every structure in every individual figure. It is also noted that these FIGS, depict portions of example systems, methods, and/or devices. However, the drawings and associated description herein can be viewed collectively and interchangeably with respect to structural components shown. Furthermore, these systems, methods, and/or devices can include other structures not shown that may be present upstream and/or downstream from the illustrated structures. For example, the structures shown can be part of a sample preparation cartridge module. The sample preparation cartridge module may include interconnected volumes arranged in series between the input and output in a linear direction to receive a vertically layered density gradient column. The various volumes may include, for example, the bulk fluid volume 232 and the capillary volume 236. However, there may be other volumes present above or below these portions, or which are included as part of these portions, e.g., sub-volumes. For example, the bulk fluid volume may include a mixing chamber 234 connected to the biological sample input to contain and mix a composition comprising a biological sample and a particulate substrate. In this example, the mixing chamber may reside as part of the bulk fluid volume separated by a displacable membrane or other barrier, e.g., piercable, puncturable, removable, etc. In other examples, the mixing chamber may reside along the entire bulk fluid volume (not shown). The capillary volume, on the other hand, may include a fluidic isolation chamber downstream of the bulk fluid volume (which may include a mixing chamber or be a mixing chamber) to separate particulate substrate and a biological component from the biological sample. The separation may be by the introduction of a non-newtonian fluid along the fluid column, or in other examples, the introduction of a gas, e.g., air bubble in the capillary volume to separate the mixing chamber from the fluidic isolation chamber, as described in greater detail hereinafter.

[0015] Turning more specifically now to FIG. 2A, a device 200 for forming a density gradient column of multiple fluids within interconnected volumes 205, such as may be carried by a vessel, e.g., a unitary or modular vessel. In this example, a sample fluid 210 can be dispensed through a fluid injection opening 220 into a bulk fluid volume 232 which in this example includes a mixing chamber 234 that can be defined temporarily by a penetrable seal 238. Below the bulk fluid volume resides a capillary volume 236. Magnetizing particles 240 are shown in the sample fluid. The sample fluid can be heated to lyse the biological sample and release the biological component of interest therein. The biological component of interest may be a nucleic acid, for example. The sample fluid can then be cooled and rotationally agitated by a rotational plunger 250 to increase collisions between the released biological component and the magnetizing particles thereby facilitating binding of the biological component to the magnetizing particles. As shown in FIG. 2B, a wash buffer 260 can be loaded into the bulk fluid volume from below through the capillary volume by depressing a wash buffer reservoir 262 that includes the wash buffer therein. Air in the density gradient column can exit through an air vent 270, for example. While not illustrated, in some examples, an air pocket may remain in the capillary volume below the wash buffer reservoir near the cap. As shown in FIG. 2C, the penetrable seal 238 can be punctured by depressing the rotational plunger 250 therethrough, thus, placing the sample fluid on top of the wash buffer fluid in the bulk fluid volume. The density of the wash buffer can be greater than that of the sample fluid, and thus, a density gradient column is formed. While the wash buffer is illustrated as extending in the bulk fluid volume, in some examples, the wash buffer can reside completely within the capillary volume and the sample fluid may extend into the capillary volume. As shown in FIG. 2D, the magnetizing particles with the biological component of interest thereon can then be magnetically moved by a magnetic field generator such as a magnet 280 or several magnets from the sample fluid into the wash buffer, and then further down into the wash buffer where the wash buffer resides with the capillary volume of the density gradient column. As shown in FIG. 2E, a portion of the wash buffer containing the magnetizing particles in the capillary volume can then be partitioned off from a balance of the wash buffer thereabove to form a partitioned sample fluid 295 that includes magnetizing particles. The partitioning can occur by depressing a gas reservoir 292 including a gas therein, such as air. The gas enters the capillary volume of the density gradient column forming a separation gas bubble 290 between the wash buffer and the partitioned sample fluid. Because of the capillary forces of the capillary volume, e.g., the small size of the capillary volume relative to the air, the air bubble created does not escape thereabove into the other fluids, but rather remains as an air bubble partition between the partitioned sample fluid and the balance of both fluids thereabove. The capillary force at an interface between the separation gas bubble and the fluid thereabove can be greater than a buoyance force of the gas if located in the fluid thereabove. As shown in FIG. 2F, a reconstitution fluid reservoir 312 that contains a reconstitution buffer can be used to mix with a dry reagent 320 to form a reconstituted fluid reagent 330, for example. Thus, the reconstitution fluid reservoir can be depressed to force the fluid into contact with the dry reagent to form the reconstituted fluid reagent which can be injected into the capillary volume adjacent to (as shown) or to mix (not shown) with the partitioned sample fluid. The gas bubble and then the reconstituted fluid reagent can both come through the same port, e.g., the fluid injected to reconstitute the dry reagent can displace the air therein into the capillary volume. However, it is noted that these two fluids (the gas bubble and the reconstituted fluid reagent) can alternatively be injected into the capillaryvolume through separate ports in other examples. In further detail, as shown in FIG. 2G and FIG. 2H, a fluid plug 340 can be formed by depressing a non-newtonian plugging fluid reservoir 342 including a non-newtonian fluid therein, thereby dispensing the non-newtonian fluid into the capillary volume of the density gradient column. A cap 350 at the end of the capillary volume can be removed, and the partitioned sample fluid, which may indude the reconstituted fluid reagent and the magnetizing partides can be dispensed into a receptacle 360 through a biological sample output 355, for example. Alternatively, in one example, as shown in FIG. 2! and FIG. 2J, the reconstituted fluid reagent, the partitioned sample fluid, and the magnetizing particles can be heated to decouple the isolated biological component from the magnetizing particles. The magnetizing particles may be held in place or moved away from the dispensing tip with a magnet and then the cap can be removed, allowing the reconstituted fluid reagent, the partitioned sample fluid, and the isolated biological component to dispense into a receptacle without the magnetizing particles. The method illustrated above exemplifies one sequential order for isolating the biological component from the biological sample. However, the methods are not so limited. For example, the sample fluid could be loaded into the interconnected volumes before the wash buffer is added thereto. The separation gas bubble may be formed before moving the magnetizing particles. The magnetizing particles can be magnetically moved by a magnetic field generator from the sample fluid into the wash buffer, and then into the separation gas bubble before moving further down into the wash buffer below the separation gas bubble in the capillary volume of the density gradient column. The cap can be removed before the reconstituted fluid reagent is dispensed into the capillary volume.

[0016] In further detail, dispensing a sample fluid over a wash buffer can occur by any technique that disposes the sample fluid over the wash buffer or the wash buffer below the sample fluid. In one example, the dispensing can include placing, pouring, injecting, pumping, expelling from a flexible blister pack, or otherwise positioning a wash buffer in a bulk fluid volume of a density gradient column and subsequently placing, pouring, injecting, pumping, or otherwise positioning a sample fluid over the wash buffer. In yet another example, the dispensing can occur by placing, pouring, injecting, pumping, or otherwise positioning a sample fluid in a bulk fluid volume of a density gradient column and subsequently loading a wash buffer from below the sample fluid into the bulk fluid volume of the density gradient column. The wash buffer may be loaded from below the sample fluid by injecting or pumping the wash buffer into the density gradient column or by expelling the wash buffer from a flexible blister pack by applying a force to the flexible blister pack to squeeze the wash buffer therefrom and into the density gradient column. The force applied to the flexible blister pack can be from 10 m-kg s² to 40 m-kg s² or from 10 m-kg s² to 20 m-kg-s², or from 20 m-kg s² to 40 m-kg-s². In yet other examples, a mixing chamber can be positioned above the bulk fluid volume of the density gradient column and can be separated from the bulk fluid volume by a piercable, puncturable, removable, erodible, or otherwise displacable seal, or by a valve, or the like. Upon piercing of the seal or opening of the valve, the sample fluid can be dispensed from the mixing region into the bulk fluid volume where the wash buffer may already be present, or may be loaded into the bulk fluid volume from below.

[0017] The sample fluid can include a biological sample or specimen with a biological component therein, or may include a biological component eluted from a biological sample. The sample may include a blood sample, a saliva sample, a mucous sample, a stool sample, or the like. In some examples, the biological sample may be lysed in the mixing chamber or the density gradient column. In yet other examples, the biological sample may be lysed prior to dispensing the sample fluid into the density gradient column. The lysing can occur by heating the biological sample, chemically lysing the biological sample, or a combination thereof. Lysing through heating can include heating the biological sample to a temperature ranging from 25°C to 100°C, from 30 °C to 100 °C, or from 25 °C to 75 °C for a time period ranging from 1 second to from 15 minutes, from 5 minutes to 10 minutes, from 1 second to 7 minutes, or from 10 minutes to 15 minutes. Chemical lysing can include admixing the sample fluid with a chemical lysis fluid. Suitable chemical lysis fluids may vary depending on the biological component and may include chemical lysis fluids that are compatible with the biological sample and will not interfere with subsequent processing of the biological component. Example chemical lysis fluids can include sodium dodecyl sulfate;

3-[(3-cholamldopropyl)dlmethylammonio]-1-proanesulphonate;

3-[(3~cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propanesulfonate; urea, guanidine, ethylenediaminetetraacetic acid (EDTA), cetyltrimethylammonium bromide (CTAB); surfactants, detergents, chaotropic agents, guanidine thiocyanate, guanidine hydrochioride, ethanol, isopropanol, other alcohols, and the like. When the lysis occurs in the density gradient column, a chemical lysis fluid can be placed, poured, injected, pumped, expelled from a flexible blister pack, or otherwise dispensed into the sample fluid. In one example, the chemical lysis fluid can be stored in a chemical lysis fluid reservoir and can be dispensed into the density gradient column by pumping the chemical lysis fluid from the chemical lysis fluid reservoir or expelling the chemical lysis fluid from a flexible blister pack. In another example, the chemical lysis fluid can be freeze dried and placed in the bulk fluid volume in powder form. A rotational plunger can be rotated to admix the sample fluid with the chemical lysis fluid. Lysing the biological sample or components thereof can release the biological component therein thereby allowing for an interaction between the biological component and the magnetizing particles.

[0018] In further detail, the sample fluid, e.g., biological sample fluid, can be prepared and/or loaded in any of a number manners. For example, the sample fluid may be prepared by combining multiple components within the bulk fluid volume, e.g., combining carrier fluid or buffer with magnetizing particles and the biological component within the bulk fluid volume (which may be or include a portion thereof that acts as a mixing chamber). Thus, the biological component may become associated with the magnetizing particles in the bulk fluid volume, or the biological component may already be associated with the magnetizing particles where they are combined with the fluid carrier or buffer within the bulk fluid volume. Alternatively, the sample fluid may first be prepared in a vial or other vessel outside of the bulk fluid volume, and then the sample fluid can be added into the bulk fluid volume over the wash buffer either before, after, or at the same time that the wash buffer is loaded, e.g., from the bottom up through the capillary volume or also loaded from the top prior to loading the sample fluid. To cite one specific example, a biological sample or specimen may be collected using a swab or other biological sample collection instrument. The biological sample may include the biological component of interest. The released or eluted biological component can then be placed in the carrier fluid or buffer where the biological component is eluted into the carrier fluid or buffer. The biological component can become associated with magnetizing particles in the vial or vessel (or even thereafter in the bulk fluid volume, in some examples). Then, the biological sample fluid that includes the eluted biological component can be loaded into the bulk fluid volume or a mixing chamber fluidically connected to or integrated as part of the bulk fluid volume. Thus, elution may occur outside of the cartridge module before sample input, or within the cartridge module where the carrier fluid or buffer is also loaded.

[0019] The magnetizing particles may either be present in the sample fluid when adding the sample fluid to the mixing chamber or the bulk fluid volume, or the magnetizing particles may be added thereto after the sample fluid is contained therein. The magnetizing particles may be placed, poured, injected, pumped, expelled from a flexible blister pack, or otherwise dispensed into the mixing chamber or the bulk fluid volume. In some examples, the chemical lysis fluid can include the magnetizing particles. The magnetizing particles may be added at from 5 pg to 100 pg, from 8 pg to 12 pg, from 5 pg to 50 pg, from 50 pg to 100 pg, from 25 pg to 75 pg, from 20 pg to 40 pg, from 80 pg to 100 pg, or from 5 pg to 15 pg.

[0020] In some examples, the method can include preparing the magnetizing particles by selectively binding a biological component to surface-activated magnetizing particles. The surface-activated magnetizing particles, as described in further detail below, can include an interactive surface group or a ligand on an exterior surface thereof that can be complimentary to the biological component. Selective binding can occur when combining the sample fluid including the biological component with the surface-activated magnetizing particles. In some examples, the combining may include admixing the sample fluid and the surface-activated magnetizing particles to increase collisions between the biological component and interactive surface groups or ligands on the exterior of the magnetizing particles.

[0021] The magnetizing particles can then be magnetically moved from the sample fluid into the wash buffer. The wash buffer can trap contaminates from the sample fluid and/or can remove contaminates from an exterior surface of the magnetizing particles. The magnetizing partides can further be magnetically moved from the wash buffer in the bulk fluid volume to a capillary volume of the density gradient column. Magnetically moving can include positioning a magnetic field generator to attract and draw the magnetizing particles. In some examples, the magnetic field generator can be a magnet. The magnet may be a ring magnet that can surround an exterior circumference of the density gradient column. In other examples, the magnet can be positioned on one side of the density gradient column. In yet other examples, the magnet can be positioned below the density gradient column. Applying the magnetic field, magnetic field motion, and/or differing magnetic field gradients can attract the magnetizing particles. In some examples, moving the magnetic field generator vertically along the column can attract and thereby move the magnetizing particles vertically. As the magnetic field generator continues to move vertically, the magnetizing particles will move vertically by a corresponding amount. In some examples moving the magnetizing particles can include adjusting a magnetic field generated by a magnetic field generator. As the magnetic field is strengthened, an attraction of the magnetizing particles towards the magnet will increase.

[0022] Once the magnetizing particles are situated in the capillary volume of the density gradient column, a portion of the wash buffer containing the magnetizing particles in the capillary volume can be partitioned off from a balance of the wash buffer in the bulk fluid volume of the density gradient column. The partitioning can occur between fluids in the capillary volume or can occur between fluids at the bottom of the bulk fluid volume and those inside the capillary volume. The partitioning can include forming a separation gas bubble, closing a valve, installing a plugging fluid, or a combination thereof. In one example, the partitioning can include dispensing gas into the capillary volume to form a separation gas bubble between the wash buffer in the bulk fluid volume and the partitioned sample fluid. The separation gas bubble can remain in the capillary volume due to a surface tension of the fluid relative to the size and material of the vessel at the capillary volume. An amount of gas sufficient to form a separation gas bubble can be an amount that spans an interior channel diameter of the capillary volume. The amount will depend on capillary shape and interior channel diameter. In some examples, the amount of gas dispensed can range from 0.1 μL to 500 μL, from 250 μL to 500 μL, from 0.1 μL to 300 μL, from 5 μL to 100 μL, from 100 μL to 300 μL, or from 300 μL to 500 μL. In some examples, the partitioning can further include dispensing a non-newtonian plugging fluid into the capillary volume to form a fluid plug. An amount of non-newtonian plugging fluid can be an amount that spans an interior channel diameter of the capillary volume. The dispensing of the gas, the non-newtonian plugging fluid, or the combination thereof can include injecting, pumping, or expelling of the gas and/or the non-newtonian plugging fluid from a reservoir or through an injection opening.

[0023] The dispensing of a fluid reagent into the capillary volume to interact with the partitioned sample fluid can include injecting, pumping, or expelling the fluid reagent from a reservoir or an injection opening into the capillary volume. In yet other examples, the dispensing of a dried reagent into the capillary volume can occur by positioning dried reagent in the capillary volume. The positioning can include placing, pouring, injecting, pumping, or expelling of the dried reagent into the capillary volume. The dried reagent may be placed in the capillary volume at any time during the method including prior to positioning the sample fluid over the wash buffer. A dried reagent can be reconstituted by the partitioned sample fluid. A type and amount of the fluid reagent or the dried reagent that can be dispensed can be based on what is appropriate for subsequent processing of the biological component and is not particularly limited. The dispensing of the fluid reagent or the dried reagent into the capillary volume can occur upstream or downstream from where the magnetizing particles are situated in the capillary volume. In one example, the dispensing can include expelling a fluid reagent from a fluid reagent reservoir. In yet other examples, the dispensing can include injecting a fluid reagent from an injection opening. In a further example, the dispensing can include expelling a dried regent from a flexible blister pack into the capillary volume. In one example, the partitioning of the magnetizing particles and the dispensing of the fluid reagent can occur sequentially by reconstituting dried reagent stored within a dry reagent reservoir. The dry reagent reservoir can also include an amount of air and can be fluidically connected to the capillary volume. Upon reconstituting of the dried reagent with a reconstituting buffer, the dried reagent can dissolve or dispense in the reconstituting buffer to form a reconstituted fluid reagent. The air in the reservoir can be forced into the capillary volume and then the reconstituted fluid reagent can be forced into the capillary volume.

[0024] In some examples, the biological component can be separated from the magnetizing particles in the capillary volume thereby releasing isolated biological component into the fluid reagent and/or the partitioned sample fluid. The separating can include heating the magnetizing particles and the fluid reagent. The heating can be at a temperature ranging from 40 °C to 95 °C, from 50 °C to 75 °C, or from 40 °C to 80 °C for a time period ranging from 1 second to 10 minutes, from 2 seconds to six minutes, from 5 minutes to 10 minutes, or from 2 minutes to 8 minutes. Releasing the biological component can allow for independent analysis of the biological component which may not be possible if the magnetizing particles would otherwise interfere with the subsequent analysis.

[0025] In some examples, the method can include dispensing the partitioned sample fluid and the fluid reagent from the capillary volume of the density gradient column. Dispensing the partitioned sample fluid and the fluid reagent can include opening a valve, removing a cap, or piercing a seal that may be preventing fluid flow out of the density gradient column. In some examples, a magnetic field may be applied to the capillary volume to trap the magnetizing particles in the capillary volume to separately dispense isolated biological component along with the partitioned sample fluid and the fluid reagent.

Systems for Isolating a Biological Components from Biological Samples

[0026] In accordance with examples of the present disclosure, a system for isolating a biological component from a biological sample is presented. The system 300 can include, magnetizing particles, a density gradient column, a dry reagent reservoir or a fluid reagent reservoir and a magnetic field generator. As illustrated in FIG. 3, the magnetizing particles 240 can be surface-activated to bind with a biological component, or can be bound to the biological component. The system can include an interconnected volumes 205 (which may be defined by a unitary vessel or a modular vessel) to hold a density gradient column with fluid of differential densities, and can include a bulk fluid volume 232 and a capillary volume 236. In some examples, the bulk fluid volume may include a mixing chamber 234. The density gradient column can be established to receive a sample fluid 210 and a wash buffer 260 having a greater density than the sample fluid. The bulk fluid volume of the density gradient column can include the sample fluid and the wash buffer therein or can be established to contain the sample fluid and the wash buffer. The dry reagent reservoir 322 can include a dried reagent 320 and can be positioned outside of the density gradient column and fluidical ly connected to the capillary volume of the density gradient column. The system may also include a magnetic field generator, such as a magnet 280 that can draw the magnetizing particles from the sample fluid into the wash buffer along the density gradient column and into the capillary volume. The magnetizing particles, the density gradient column, the dry reagent reservoir, and the magnetic field generator can be as described below. In some examples, the system can further include a biological sample input 220 and a biological sample output 355, for example. Other compounds, fluids, and/or structures that may be present were described by example in FIGS. 2A-2J, including a fluid reagent(s) or a dried reagent(s), a valve(s), a vent(s), a cap at the biological sample output, a receptacle for accepting the partitioned sample fluid (with or without reagent and/or magnetizing particles), etc. Some of these compounds and structures are described in greater detail hereinafter.

Density Gradient Columns

[0027] The density gradient columns described herein can be carried byinterconnected volumes that include a bulk fluid volume and a capillary volume. The interconnected volumes can be defined, for example, by a unitary or a modular vessel. The bulk fluid volume can be upstream of the capillary volume. The bulk fluid volume can have a larger cross-section than a cross-section of the capillary volume. In some examples, the bulk fluid volume may include a mixing chamber. The bulk fluid volume can indude a conical portion or tapered portion that connects the bulk fluid volume to the capillary volume. A cross-section of the chamber can be round, square, triangle, rectangle, or other polygonal in shape. In some examples, the bulk fluid volume can have a diameter (or width if not circular) at the widest cross-section of from 3 mm to 20 mm, from 5 mm to 15 mm, from 3 mm to 12 mm, from 10 mm to 20 mm, or from 3 mm to 10 mm. The bulk fluid volume can be where a majority of the fluid in the density gradient column resides, for example. The bulk fluid volume can connect to the capillary volume at a capillary junction. In the bulk fluid volume, the density gradient column can be maintained where multiple fluids interface one another by the density differential of the relative fluids in contact with one another. For example, a fluid with a higher density can reside beneath a fluid with a lower density, and they may remain separated due to their density differential, even without barriers therebetween.

[0028] The capillary volume can have a smaller cross-section than a cross-section of the bulk fluid volume. The capillary volume can be an elongated tubular region and can have a round, square, triangle, rectangle, or other polygonal cross-section. In some examples, the capillary volume at the widest cross-section can have an interior opening diameter of from 0.1 mm to 4 mm, from 0.1 mm to 2 mm, from 0.5 to 1.5 mm, from 1 mm to 3 mm, from 2 mm to 4 mm, or from 2 mm to 3 mm. The capillary volume may be tapered. For example, the capillary can be tapered and can have an interior channel diameter of 4 mm at one end to an interior channel diameter at the opposite end of 1 mm, or the capillary can be tapered from an interior channel diameter of 3 mm at one end to an interior channel diameter at the opposite end of 1 mm, or from 2 mm at one end to an interior channel diameter at the opposite end of 1 .5 mm, or from 2 mm at one end to an interior channel diameter at the opposite end of 1 mm. Notably, within the capillary volume, the density gradient column may or may not have fluids along the column separated strictly by their density. Because of the narrow passageway, the capillary forces provided by the interaction of the fluids, the fluid interfaces, and the narrow passageway enables in some instances lower density fluids to be retained beneath higher density fluids. For example, even a gas may be able to be retained within the capillary volume below higher density liquid fluids due to the capillary forces at work.

[0029] The density gradient column within the interconnected volumes can be contained by a vessel made of various polymers (e.g. Polypropylene, TYGON, PTFE, COC, others), glass (e.g. borosilicate), metal (e.g. stainless steel), or a combination of materials. Additionally, the capillary component could be formed from multiple materials used in various microfluidic devices, such as silicon, glass, SU-8, ROMS, a glass slide, a molded fluidic channel(s), 3-D printed material, and/or cut/etched or otherwise formed features. The density gradient column may be monolithic or may be a combination of components fitted together.

[0030] The vessel can be shaped and/or configured, etc., to receive fluids, such as a sample fluid, a lysis buffer, a wash buffer, a gas, a reconstituted fluid reagent, and the like. Fluids can be arranged in the density gradient column in layers and individual layers can be phase separated from one another at fluid interfaces. In some examples, the phase separation can be based on fluidic properties of the various fluids, including a density of the respective fluids along the column. Fluid layers can be in fluid communication with adjoining fluid layers.

[0031] In the bulk fluid volume, the greater or higher the density of a fluid, relative to other fluids in the column, the closer to the bottom of the bulk fluid volume the fluid will be located. For example, when arranged vertically a first fluid having a first density can form a first layer of the density gradient column. A second fluid having a second density greater than a density of the first fluid can form a second fluid layer of the density gradient column. A third fluid having a third density greater than a density of the second fluid can form a third fluid layer of the density gradient column and the like. In one example, the density gradient column can include a sample fluid positioned on top of a wash buffer, where the wash buffer has a greater density than the sample fluid.

[0032] A density of a fluid can be altered using a densifier. Example densifiers can include sucrose, cesium based densifiers such as CsCI, 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.

[0033] In the capillary volume, a surface tension of the fluid relative to the size and material of the vessel (defining the interconnected volumes) can provide the ability to position less dense fluids beneath fluids of greater density. In some examples, a separation gas bubble can be formed in the capillary volume of the density gradient column. The separation gas bubble can become trapped in the capillary volume due to the surface tension in the capillary volume. A fluid having a density less than a density of fluids above the separation gas bubble can be located below the separation gas bubble. For example, the fluid that is above the separation gas bubble can include densifiers, as described above, and the fluid below the gas bubble can be free of densifiers so that the fluid above the separation gas bubble has a higher density. In various examples, the density difference between the fluid above the separation gas bubble and the fluid below the separation gas bubble 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. In one example, a reconstituted fluid reagent including the dried reagent and a reconstitution buffer may be positioned in the capillary volume of the density gradient column below the separation gas bubble. The separation gas bubble can prevent intermixing despite the density difference.

[0034] A quantity of fluid layers in the density gradient column is not particularly limited. In one example, the density gradient column can include a sample fluid and a wash buffer. In another example, the density gradient column can include a sample fluid, a wash buffer, air, and a fluid reagent. In yet another example, the density gradient column can include a sample fluid, a lysis buffer, a wash buffer, air, and a fluid reagent.

[0035] The fluid layers in the density gradient column can be formulated to interact with the magnetizing particles. Individual fluid layers can have a different function. For example, a fluid layer can include a lysis buffer to lyse cells. In yet other examples, a fluid layer can be a surface binding fluid layer to bind the biological component to the magnetizing particles, a wash fluid layer can trap contaminates from a sample fluid and/or remove contaminates from an exterior surface of the magnetizing particles, a surfactant fluid layer can coat the magnetizing particles, an elution fluid layer can 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), and so on. In some examples, individual fluid layers can provide sequential processing of a biological sample.

[0036] A vertical height of individual fluid layers in the density gradient column can vary. Adjusting a vertical height of an individual fluid layer can affect a residence time of the magnetizing particles in that fluid layer. The taller the fluid layer, the longer the residence time of the magnetizing particles in the fluid layer. In some examples, all of the fluid layers in the density gradient column can be the same vertical height. In other examples, a vertical height of individual fluid layers in a multi-fluid density gradient column can vary from one fluid layer to the next. In one example, a vertical height of the individual fluid layers can individually range from 10 pm to 50 mm. In another example, a vertical height of the individual fluid layers can individually range from 10 pm to 30 mm, from 25 pm to 1 mm, from 200 pm to 800 pm, or from 1 mm to 50 mm.

[0037] The vessel that defines the interconnected volumes and carries the density gradient column may further include one or more openings, inputs, outputs, and/or ports. For example, the vessel may include an opening, an input, and/or a port to permit loading of fluids and reagents into the density gradient column. For example, a fluid injection opening can permit loading of a sample fluid, a wash buffer, and the like into the bulk fluid volume of the density gradient column. The vessel may also include one or more outputs, such as a biological sample output as described previously. For example, the capillary volume may include a fluidic output that can permit dispensing of a biological component, a biological sample, a fluid, magnetizing particles, or a combination thereof from the density gradient column. In yet other examples, the vessel may include an output for venting gas to relieve pressure in the density gradient column. [0038] In yet other examples, the bulk fluid volume, the capillary volume, or a combination thereof can include seals, valves, plugs, or a combination thereof. In one example, seals, valves, and/or plugs can be used to temporarily prevent mixing of fluids and allow for independent manipulation of fluids. For example, a mixing chamber can be positioned as part of the bulk fluid volume or can be positioned above the bulk fluid volume of the density gradient column. The bulk fluid volume of the density gradient column can be separated from the mixing chamber by a displacable seal. The seal can allow for independent manipulation of the sample fluid before the sample fluid may be positioned over the wash buffer. In yet another example, a fluid plug can be formed in the capillary volume of the density gradient column. The fluid plug may hold back pressure in the density gradient column and can thereby allow dispensing of a downstream portion of fluid which can be located beneath the fluid plug, while preventing dispensing of fluid upstream of the fluid plug.

[0039] The vessel that defines the interconnected volumes can also be shaped or configured to be coupled with a removable or puncturable cap or seal that can cover an opening of the density gradient column vessel. The vessel can be a standalone component or can be part of a larger device or system that includes fluids or other components connected in series or parallel, for example.

Magnetizing Partides

[0040] The magnetizing particles, in further detail, can be in the form of paramagnetic particles, superparamagnetic particles, diamagnetic particles, or a combination thereof, for example. The term “magnetizing particles” is defined herein to include particles or microparticles, e.g., magnetizing 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 field is increased, or the magnetizing particles get closer to a magnet applying the magnetic field. [0041] In more specific detail, “paramagnetic particles” have these properties, in that they have the ability to increase in magnetism when a magnetic field is present; however, paramagnetic particles are not magnetic when a magnetic field is not present. In some examples, the paramagnetic particles can exhibit no residual magnetism once the magnetic field is removed. A strength of magnetism of the paramagnetic particles can depend on the strength of the magnetic field, the distance between a source of the magnetic field and the paramagnetic particles, and a size of the paramagnetic particles. As a strength of the magnetic field increases and/or a size of the paramagnetic particles increases, the strength of the magnetism of the paramagnetic particles increases. As a distance between a source of the magnetic field and the paramagnetic particles increases, the strength of the magnetism of the paramagnetic particles decreases. “Superparamagnetic particles” can act similar to paramagnetic particles; however, they can exhibit magnetic susceptibility to a greater extent than paramagnetic particles in that the time it takes for them to become magnetized appears to be near zero seconds. “Diamagnetic particles,” on the other hand, can display magnetism due to a change in the orbital motion of electrons in the presence of a magnetic field.

[0042] The magnetizing particles can be surface-activated to selectively bind with a biological component or can be bound to a biological component from a biological sample. An exterior of the magnetizing particles can be surface-activated with interactive surface groups that can interact with a biological component of a biological sample or may include a covalently attached ligand. In some examples, the ligand can include proteins, antibodies, antigens, nucleic acid primers, nucleic acid probes, amino groups, carboxyl groups, epoxy groups, tosyl groups, sulphydryl groups, or the like. In one example, the ligand can be a nucleic acid probe. The ligand can be selected to correspond with and to bind with the biological component. The ligand may vary based on the type of biological component targeted for isolation from the biological sample. For example, the ligand can include a nucleic acid probe 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. In one example, the magnetizing particles can be surface-activated to bind to nucieic acid such as DNA or RNA. Thus DNA or RNA molecules can be bound to the surface of the magnetizing particles. Commercially available examples of magnetizing particles that are surface-activated include those sold under the trade name DYNABEADS®, available from ThermoFischer Scientific (USA).

[0043] In some examples, the magnetizing particles can have an average particle size that can range from 10 nm to 50,000 nm. In yet other examples, the magnetizing particles can have an average particle size that can range from 500 nm to 25,000 nm, from 10 nm to 1 ,000 nm, from 25,000 nm to 50,000 nm, or from 10 nm to 5,000 nm. The term “average particle size" describes a diameter or average diameter, which may vary, depending upon the morphology of the individual particle. A shape of the magnetizing particles 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 magnetizing particles 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.

[0044] In one example, the magnetizing particles can be unbound to a biological component when added to the density gradient column. Binding between the magnetizing particles and the biological component of the biological sample can occur within the density gradient column. In yet another example, the magnetizing particles and a biological sample including a biological component can be combined before the sample fluid is added to the density gradient column. Magnetic Field Generators

[0045] The method and system herein can further include a magnetic field generator capable of generating a magnetic field. The magnetic field may be turned on and off by introducing electrical current/voltage to the magnet. The magnetic field generator can be permanently placed, can be movable along the density gradient column, or can be movable in position and/or out of position to effect movement of the magnetizing particles in and through the density gradient column.

[0046] The magnetizing particles can be magnetized by the magnetic field generated by the magnetic field generator. The magnetic field generator can also create a force capable of pulling the magnetizing particles through the density gradient column, holding the magnetizing particles at a location in the density gradient column, or a combination thereof. When the magnetic field generator is turned off or not in appropriate proximity, the magnetizing particles can reside In a fluid layer until gravity pulls the magnetizing particles through fluid layers of the density gradient column, 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, a quantity of the magnetizing particles, a size of the magnetizing particles, a density of the fluid in the fluid layer, a viscosity of the fluid in the fluid layer, and a surface tension at the fluid interface between fluid layers. The magnetic field generator can cause the magnetizing particles to move from one fluid layer to another, or can increase a rate at which the magnetizing particles pass from one fluid layer into another.

[0047] A strength of the magnetic field and the location of the magnetic field generator in relation to the magnetizing particles can also affect a rate at which the magnetizing particles move through the density gradient column, e.g. as the distance from the magnetizing particles increases the force applied to the magnetizing particles decreases. The further away the magnetic field generator and the lower the strength of the magnetic field, the slower the magnetizing particles will move. [0048] In an example, the magnetic field generator can be moveable in position, out of position, or at variable positions to effect downward movement, rate of movement, or to promote little to no movement of the magnetizing particles. In another example, the magnetic field generator can be positioned adjacent to a side of the multi-fluid density gradient column and can move vertically to cause the magnetizing particles to move therewith. The magnetic field generator can include a current carrying wire, a magnet, a ring magnet or the like. In an example, the magnetic field generator can be a current carrying wire. In another example, the magnetic field generator can be a ring magnet that can be placed around a circumference of the density gradient column. In yet another example, the magnetic field generator can be a magnet. In a further example, the magnetic field generator can include multiple magnets. A movable magnet(s) can likewise be positioned adjacent to a side of the multi-fluid density gradient column that is not a ring shape, but can be any shape effective for moving magnetizing particles along the density gradient column. In some examples, the magnet can be moved along a side and/or along a bottom of the multi-fluid density gradient column to pull the magnetizing particles in one direction or another. In one example, the magnet can be used to pull the magnetizing particles downwardly through fluid layers of the density gradient column.

[0049] In yet other examples, a magnetic field generator can be used to concentrate and hold the magnetizing particles near a side wall of the density gradient column. For example, the magnetic field generator can concentrate the magnetizing particles near a side wall of the density gradient column and heat can be applied to decouple and separate an isolated biological component from the magnetizing particles. The magnetic field generator can continue to hold the magnetizing particles while an outlet of the density gradient column can be opened thereby allowing dispensing of the isolated biological component from the density gradient column separate of the magnetizing particles. Reservoirs

[0050] In some examples, reservoirs can be included for holding and dispensing fluids and reagents utilized in the methods and incorporated with the systems disclosed herein. Reservoirs can be positioned outside of the interconnected volume which contains or is adapted to contain the density gradient column. In some examples, the reservoirs can be fluidically connected to the interconnected volumes via an opening, a microchannel, or an inlet to permit dispensing of a content within the reservoir into the density gradient column.

[0051] Reservoirs can vary in type. For example, a reservoir can be a chamber, a channel, a flexible blister pack, or a combination thereof. In one example, a reservoir can be a flexible blister pack that when pushed, can open and force contents within out of the reservoir and into the density gradient column. In some examples, the reservoir can include a sealing layer that can maintain separation of contents in the reservoir and the density gradient column until the sealing layer is broken. Breaking the sealing layer may allow contents of the reservoir to be released therefrom.

[0052] Reservoirs can be sized and shaped to contain a fluid, a reagent, or a combination thereof. Types of reservoirs can include a chemical lysis fluid reservoir, a wash buffer reservoir, a gas reservoir, a dry reagent reservoir, a non-newtonian plugging fluid reservoir, a reconstitution buffer reservoir, a fluid reagent reservoir, or a combination thereof.

[0053] A chemical lysis fluid reservoir, in further detail, can be sized and shaped to contain a chemical lysis fluid. In some examples, the lysis buffer reservoir can include the chemical lysis fluid therein. The chemical lysis fluid may be as previously discussed. The chemical lysis fluid reservoir may be located to allow dispensing of the chemical lysis fluid into the sample fluid when positioned along the density gradient column.

[0054] A wash buffer reservoir, in further detail, can be sized and shaped to contain a wash buffer. In some examples, the wash buffer reservoir can include a wash buffer therein. The wash buffer can be an aqueous solution. For example, a wash buffer can include water, alcohol (such as ethanol), a binding agent, a salt, a surfactant, a stabilizing agent, buffering agents to maintain pH, or a combination thereof. In some examples, the wash buffer can include a densifier. Any fragments and other materials from the biological sample that may be adhere to the magnetizing particles at locations other than the interactive surface group or the ligand on the exterior surface thereof can be washed off by the wash buffer. Thus, the wash buffer can be a liquid that can wash off these materials while also being safe for the biological component. The wash buffer reservoir may be located to allow dispensing of the wash buffer into the density gradient column.

[0055] A gas reservoir can be sized and shaped to contain a gas in an amount capable of forming a separation gas bubble in the capillary volume of the density gradient column. The gas reservoir may include a gas, such as air. The gas reservoir may be located to allow dispensing of a gas into the capillary volume of the density gradient column.

[0056] A dry reagent reservoir can be sized and shaped to contain a dried reagent. In one example, the dry reagent reservoir can include the dried reagent. In some examples, a dried reagent can includes all ingredients for analyzing a sample other than water. To enable release of the dried reagent from the dry reagent reservoir, the dry reagent reservoir can either include a reconstitution buffer injection opening, can be in fluidic connection with a reconstitution buffer reservoir positioned upstream of the dry reagent reservoir, or can include a combination thereof. A reconstitution buffer injection opening and/or a reconstitution buffer reservoir can allow a reconstitution buffer to be added to the dried reagent to form a reconstituted fluid reagent. The reconstituted fluid reagent can be dispensed into the capillary volume of the density gradient column.

[0057] A reconstitution buffer reservoir can be sized and shaped to contain a reconstitution buffer. In some examples, the reconstitution buffer reservoir can include a reconstitution buffer. The reconstitution buffer can be any aqueous solvent. In one example the reconstitution buffer can be water. In other examples, the reconstitution buffer can include additional ingredients, such as salts, surfactants, buffering agents to maintain pH, and others. The reconstitution buffer reservoir, as previously discussed, can be arranged to allow dispensing of the reconstitution buffer into a dry reagent reservoir. [0058] A fluid reagent reservoir can be sized and shaped to contain a fluid reagent. In some examples, the fluid reagent reservoir can include a fluid reagent. The fluid reagent is not particularly limited and may depend on the analysis to be performed. In one example, the fluid reagent can be a master mix. The fluid reagent reservoir can be arranged to allow dispensing of the fluid reagent into the capillary volume of the density gradient column.

[0059] The dry reagent and/or the fluid reagent can include a reactant useful to mix with a biological component for further analysis. In one example, reactant can be selected from PCR master mix, nucleic acid primers, deoxynucleosides, triphosphates, reverse transcriptase, secondary antibodies, polymerases, enzymes, polymerases, probes, magnesium salt, bovine serum albumin (BSA), beads, or a combination thereof. PCR master mix can include a mixture of multiple compounds that are used in a PCR assay. These compounds can include DNA polymerase, nucleoside triphosphate, deoxyribose nucleoside triphosphate, magnesium chloride, magnesium sulfate, template DNA, forward primer, reverse primer, tris hydrochloride, potassium chloride, and others. In certain examples, the reactant can be a lyophilized PCR master mix. Examples of commercially available PCR master mixes can include TITANIUM TAQ ECODRY™ premix, ADVANTAGE 2 ECODRY™ premix (available from Takara Bio, Inc. Japan); Lyophilized Ready-to-Use and Load PCR Master Mix (available from Kerafast, Inc., USA); MAXIMO™ Dry-Master Mix (available from GenEon Technologies, USA), and others.

[0060] A non-newtonian plugging fluid reservoir can be sized and shaped to contain a non-newtonian plugging fluid. In some examples the non-newtonian plugging fluid reservoir can include a non-newtonian plugging fluid. Non-newtonian plugging fluids can include a Bingham plastic, a viscoplastic, or a shear thinning fluid. Bingham plastics can include materials that behave as rigid bodies at low stress but which flow as a viscous fluid at high stress. The transition between the rigid body behavior and the viscous fluid behavior can occur at various different stress levels, depending on the particular Bingham plastic material. Bingham plastics can include greases, slurries, suspensions of pigments, and others. Viscoplastics are a broader category of materials that can include Bingham plastics. Viscoplastic materials can experience irreversible plastic deformation when stress over a certain level is applied. When stress under this level is applied, the viscoplastic material can behave as a rigid body, as is the case with Bingham plastics, or the viscoplastic material can undergo reversible elastic deformation. Shear thinning fluids are materials that behave as a fluid with a high viscosity when low stress is applied, but the viscosity of the fluid decreases when the stress is increased. Examples of shear thinning fluids can include polymer solutions, molten polymers, suspensions, colloids, and others. In one example, the non-newtonian plugging fluid can include a mineral oil-based grease, a vegetable oil-based grease, a petroleum oil-based grease, a synthetic oil-based grease, a semi-synthetic oil-based grease, a silicone oil-based grease, or a combination thereof. The plugging fluid reservoir may be located to allow dispensing of the non-newtonian plugging fluid into the capillary volume of the density gradient column.

[0061] The viscosity of the non-newtonian plugging fluid can be sufficient to separate fluids above the plug of non-newtonian plugging fluid from fluids below the plug of non-newtonian. This can include holding a pressure head of the fluids above the non-newtonian fluid when the fluid column is oriented vertically. In some examples, the viscosity of the non-newtonian plugging fluid can be effectively infinite up to a threshold stress. In these examples, the non-newtonian plugging fluid can act as a rigid body when the stress on the fluid is below the threshold. In other examples, the non-newtonian plugging fluid can have a viscosity that is sufficient to support the fluids above the plug for an amount of time that can allow fluid below the plug to be ejected from the device without mixing the fluid above the plug. In certain examples, the non-newtonian fluid plug can have a viscosity of greater than 5,000 centipoise, or greater than 10,000 centipoise, or greater than 15,000 centipoise, or greater than 20,000 centipoise.

[0062] Reservoirs may be arranged to allow a fluid or a reagent therein to be individually dispensed into the density gradient column; can be arranged in series to allow a fluid, a reagent, or a combination thereof to be dispensed sequentially or at the same time into the density gradient column; or can be arranged to allow for a combination thereof.

Definitions

[0063] 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.

[0064] As used herein, “Bingham plastic" refers to a class of materials that behave as rigid bodies at low stress but which flow as a viscous fluid at high stress. The transition between the rigid body behavior and the viscous fluid behavior can occur at various different stress levels, depending on the particular Bingham plastic material. Bingham plastics can include greases, slurries, suspensions of pigments, and others.

[0065] As used herein, the term “interact" as it relates to a surface of the magnetizing particles indicates that a chemical, physical, or electrical interaction occurs where the magnetizing particles surface properties that are different may have been present prior to entering the fluid layer, but does not include modification of properties of the bulk of the 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. In yet other examples, a fluid layer can be a surface binding fluid layer to bind the biological component to the magnetizing particles, 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, 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 PGR, and so on. [0066] As used herein, “viscoplastic” refers to a broader category of materials that can include Bingham plastics. Viscoplastic materials can experience irreversible plastic deformation when stress over a certain level is applied. When stress under this level is applied, the viscoplastic material can behave as a rigid body, as is the case with Bingham plastics, or the viscoplastic material can undergo reversible elastic deformation.

[0067] 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 are 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.

[0068] 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.

EXAMPLES

[0069] The following illustrates several examples of the present disclosure. However, the following are illustrative of the application of the principles of the present disclosure. Numerous modifications and alternative compositions, methods, and systems may be devised without departing from the spirit and scope of the present disciosure. The appended claims are intended to cover such modifications and arrangements.

Example 1 - RNA Isolation

[0070] A biological sample from saliva was gathered on a collection swab. The collection swab was placed in a 3 mL buffer solution of Tris HCL, magnesium salts, and surfactant to prepare a sample fluid. Eight to twelve pg of magnetizing particles including silica, an iron core, and surface activation groups of nucleic acid probe complimentary to a selected stand of RNA were added to the sample fluid. The magnetizing particles had an average particle size of 1 micron. The sample fluid was then placed in a mixing chamber situated over a bulk fluid volume of a density gradient column. The mixing chamber and the bulk fluid volume were separated by a pierce-able seal. To lyse cells of the biological sample, the sample fluid in the mixing chamber was heated to a temperature of 80 °C for a time period of three minutes. The sample fluid was then agitated using an instrument that spun the sample fluid, allowing the RNA from the lysed cells to bind with nucleic acid probes on the magnetizing particles. One hundred mLs of a wash buffer stored in a wash buffer reservoir including a foil blister was added to a bulk fluid volume of the density gradient column from below the density gradient column by compressing the foil blister. The seal separating the mixing chamber from the bulk fluid volume including the wash buffer therein was pierced, which allowed the sample fluid to become positioned over the wash buffer in the bulk fluid volume. The magnetizing particles were then transported from the sample fluid, through the wash buffer, and into a capillary volume of the density gradient column by two magnets, located on opposing sides of the interconnected volumes. As the magnetizing particles passed through the wash buffer the magnetizing particles were purified of contaminates and a portion of the wash buffer was pulled into the capillary volume along with the magnetizing particles. A reconstitution butter reservoir including a foil blister was pierced allowing a reconstitution buffer to flow through a microchannel and into a dry reagent reservoir including a dry reagent and air. As the reconstitution buffer entered the dry reagent reservoir, air in the dry reagent reservoir was forced upward into the capillary volume of the density gradient column followed by a reconstituted fluid reagent that included the dry reagent and the reconstituted buffer. The air created a separation gas bubble between the portion of the wash buffer in the bulk fluid volume and the portion of the wash buffer that passed into the capillary volume of the density gradient column along with the magnetizing particles. The reconstituted fluid reagent was allowed to admix with the portion of the wash buffer that passed into the capillary volume of the density gradient column along with the magnetizing particles to form a partitioned sample fluid. An entrance to the capillaryvolume was sealed by puncturing a non-newtonian plugging fluid reservoir. A non-newtonian plugging fluid was then expelled into the capillary volume to form a fluid plug between the portion of the wash buffer above the partitioned sample fluid and the partitioned sample fluid. An outlet at an opposing end of the capillary volume was unsealed by a needle and the partitioned sample fluid was expelled therefrom into a collection receptacle below. The partitioned sample fluid included the reconstituted fluid reagent, the magnetizing particles with the biological component bound thereto, and the portion of the wash buffer pulled through with the magnetizing particles.

Example 2 - RNA Isolation

[0071] An RNA strand was isolated from a biological sample collected from a nasal swab, as Indicated in Example 1 . However, after sealing the capillary volume with the fluid plug, the area of the capillary volume including the partitioned sample fluid was heated to a temperature of 60 °C for a time period of 1 minute to 2 minutes to decouple the isolated biological component from the magnetizing particles. A magnet was applied to the partitioned sample fluid in the capillary volume, holding the magnetizing particles along a sidewall of the capillary volume. An outlet of the capillary volume was unsealed by a needle and the partitioned sample fluid was expelled therefrom into a collection receptacle below. The partitioned sample fluid included the reconstituted fluid reagent, the isolated biological component, and the portion of the wash buffer. The magnetizing particles remained on the sidewall of the capillary volume of the density gradient column until the magnetic field from the magnet was released.

[0072] 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 may be limited only by the scope of the following claims.

Claims

CLAIMS What is Claimed Is:

1 . A method of isolating a biological component from a biological sample, comprising: dispensing a sample fluid including magnetizing particles and a wash buffer into interconnected volumes including a bulk fluid volume fluidically connected in series with a capillary volume to form a density gradient column, the wash buffer having a greater density and positioned beneath the sample fluid within the interconnected volumes, wherein the density gradient column includes magnetizing particles; magnetically moving the magnetizing particles from the sample fluid into the wash buffer at a location residing within the capillary volume; partitioning a downstream portion of the wash buffer containing the magnetizing particles in the capillary volume from a balance of the wash buffer thereabove to form a partitioned sample fluid that includes the magnetizing particles; and dispensing a fluid reagent or a dried reagent into the capillary volume to interact with the partitioned sample fluid.

2. The method of claim 1 , further comprising preparing the magnetizing particles by selectively binding a biological component to surface-activated magnetizing particles, wherein the surface-activated magnetizing particles includes interactive surface groups or a ligand thereon complimentary to the biological component.

3. The method of claim 2, further comprising lysing a biological sample to release the biological component that was contained therein, wherein the lysing includes heating the biological sample, chemically lysing the biological sample, or a combination thereof.

4. The method of claim 2, further comprising admixing the biological component released from the biological sample with the magnetizing particles to bind the biological component to interactive surface groups or ligands on the magnetizing particles.

5. The method of claim 1 , wherein the partitioning includes dispensing a gas into the capillary volume to form a separation gas bubble between the portion of the wash buffer above the partitioned sample fluid and the partitioned sample fluid.

6. The method of claim 1 , wherein the partitioning includes dispensing a non-newtonian plugging fluid into the capillary volume to form a fluid plug.

7. The method of claim 1 , further comprising dispensing the partitioned sample fluid combined with the fluid reagent or a dried reagent that has been reconstituted from the capillary volume through an output channel.

8. The method of claim 7, wherein the partitioned sample fluid includes the biological component bound to magnetizing particles during the dispensing from the capillary volume.

9. The method of claim 1 , wherein the magnetizing particles have the biological component bound thereto and wherein the method further comprises heating the magnetizing particles and the fluid reagent in the capillary volume to separate the biological component from the magnetizing particles so that the biological component can be dispensed without the magnetizing particles.

10. The method of claim 9, further comprising applying a magnetic field to the capillary volume to trap the magnetizing particles therein, and dispensing the separated biological component from the capillary volume through an output channel.

11 . The method of ciaim 1 , wherein the partitioning of the magnetizing particies and the dispensing of the dried reagent comprises reconstituting the dried reagent stored within a dry reagent reservoir prior to dispensing into the capillary volume.

12. The method of claim 1 , wherein the partitioning of the magnetizing particles and the dispensing of the fluid reagent or dried reagent further includes forcing air from a dry reagent reservoir into the capillary volume by introducing a reconstitution fluid into a dry reagent reservoir containing the dry reagent.

13. The method of claim 1 , wherein the dispensing of the fluid reagent into the capillary volume occurs downstream from where the magnetizing particles are partitioned in the partitioned sample fluid.

14. The method of claim 1 , wherein the magnetizing particles are present in the sample fluid when dispensing the sample fluid.

15. The method of claim 1 , wherein dispensing the sample fluid and the wash buffer includes dispensing the wash buffer into the capillary volume and upward into a bulk fluid volume positioned thereabove, and dispensing the sample fluid over the wash buffer to form the density gradient column.

16. A system for isolating a biological component from a biological sample, comprising: magnetizing particles that are surface-activated to bind with a biological component, or which are bound to the biological component; interconnected volumes to receive or which contain a density gradient column including a sample fluid positioned or positionable above a wash buffer, the wash buffer having a greater density than the sample fluid, the interconnected volumes including a bulk fluid volume positioned in series with a capillary volume; a dry reagent reservoir including a dried reagent or a fluid reagent reservoir including a fluid reagent, wherein the dry reagent reservoir or the fluid reagent reservoir is positioned outside and fluidically connected to the capillary volume; and a magnetic field generator to draw the magnetizing particles along the density gradient column from the sample fluid into the wash buffer contained within the capillary volume

17. The system of claim 16, wherein the dry reagent or the fluid reagent includes PCR master mix, nucleic acid primers, deoxynucleosides, triphosphates, reverse transcriptase, secondary antibodies, polymerases, enzymes, polymerases, probes, magnesium salt, bovine serum albumin (BSA), beads, or a combination thereof.

18. The system of claim 16, further comprising a wash buffer reservoir, an air reservoir, a non-newtonian plugging fluid reservoir, a reconstitution buffer reservoir, or a combination thereof located outside of the interconnected volumes and in fluidic connection to with the density gradient column.

19. The system of claim 16, wherein the dry reagent reservoir is positioned to release a reconstituted fluid reagent including the dry reagent and a reconstitution buffer into the capillary volume beneath the wash buffer, and the reconstituted fluid reagent has a density less than the wash buffer.

20. The system of claim 16, further comprising a removable cap coupled to the end of the capillary volume at a biological sample output.