Magnetic particle air transfer

Abstract

The methods for sample preparation disclosed herein utilize an air phase to reduce the aqueous phase associated with the magnetic particles bound to the nucleic acids, reducing the carryover of one or more contaminants, such as cell debris, chaotropic agents, non-specifically attached molecules, etc. This air transfer step is improved by using a combination of a first population of magnetic particles and a second population of magnetic particles, where the first population of magnetic particles is capable of associating with the nucleic acids, and the magnetic particles in the second population are at least two times larger in size than the magnetic particles in the first population. As discussed herein, the use of this second population of magnetic particles improves the transfer of the first population of magnetic particles by reducing the loss of the first magnetic particles during transfer. The sample preparation method can be semi-automated or fully automated.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 143,494, filed on 29 January 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Introduction Nucleic acid isolation and purification is a set of molecular biological techniques used to extract DNA and RNA for use in downstream applications. Nucleic acid isolation and purification methods include column-based isolation and purification, reagent-based isolation and purification, magnetic bead-based isolation and purification, and other techniques. Reagents, kits, and instruments used for nucleic acid isolation and purification are available. Inadequate sample preparation can lead to suboptimal results in downstream applications; therefore, optimized versions of kits have emerged to address variations in sample sources such as blood, plant tissue, fungi, or bacteria.

[0003] The sample preparation process includes releasing nucleic acid targets from their natural biosource using chaotropic nucleic acid extraction techniques (e.g., lysing cells such as patient cells, or lysing microorganisms such as viruses, bacteria, and fungi), binding the nucleic acids to a solid phase (e.g., paramagnetic particles) using silica or iron oxide nucleic acid chemistry, separating the solid phase from the residual lysis solution using magnetic separation techniques, washing to remove unwanted materials, and eluting or separating the nucleic acids from the solid phase using fluid handling techniques. Upon completion of the sample preparation protocol, the sample is transferred to the PCR component of the device for nucleic acid detection. [Overview of the project] [Means for solving the problem]

[0004] Aspects of this disclosure include a sample preparation method, a sample preparation cartridge, and a sample preparation system.

[0005] A method for sample preparation disclosed herein utilizes a step of transferring magnetic particles bound to the analyte through an air phase. This transfer step is referred to as air transfer. The air transfer step reduces carryover of the aqueous phase by the magnetic particles. Reducing carryover of the aqueous phase by the magnetic particles reduces contaminants such as cell debris, chaotropic agents, and nonspecifically attached molecules. This air transfer step is improved by using a combination of a first population of magnetic particles and a second population of magnetic particles, wherein the first population of magnetic particles is capable of associating with the target analyte, and the magnetic particles in the second population are at least twice as large in size as the magnetic particles in the first population. As considered herein, the use of this second population of magnetic particles improves the transfer of the first population of magnetic particles by reducing the loss of the first magnetic particles during air transfer. The sample preparation method may be semi-automatic or fully automated.

[0006] Also provided herein is a sample preparation cartridge comprising a first chamber containing a first collection of magnetic particles and a second collection of magnetic particles, and a second chamber which can be configured as an air chamber and is adjacent to the first chamber, wherein the magnetic particles may optionally be in pellet form or optionally in freeze-dried form.

[0007] A system for sample preparation provided herein includes a disclosed sample preparation cartridge and a magnet operably positioned in relation to the sample preparation cartridge such that the magnet can exert a magnetic force on magnetic particles present in the sample preparation cartridge. The system may optionally include an instrument comprising a processor that includes instructions for performing one or more steps of the disclosed method. [Brief explanation of the drawing]

[0008] [Figure 1A] A schematic diagram of the sample preparation cartridge and reagents for carrying out the disclosed method is shown. [Figure 1B]A schematic diagram of the sample preparation cartridge and reagents for carrying out the disclosed method is shown. [Figure 1C] Figure 1D shows a sample preparation cartridge having three connected chambers. An air chamber is positioned between two chambers containing the aqueous phase. A magnet is shown operably positioned adjacent to the first chamber. [Figure 1D] Figure 1D shows a sample preparation cartridge having three connected chambers. An air chamber is positioned between two chambers containing the aqueous phase. A magnet is shown operably positioned adjacent to the first chamber. [Figure 2] A schematic diagram of a sample preparation cartridge and a method step for sample preparation according to one embodiment of the present disclosure is shown. [Figure 3] A schematic diagram of a sample preparation cartridge and a method step for sample preparation according to another embodiment of the present disclosure is shown. [Figure 4A] An embodiment of the present disclosure shows a cylindrical sample preparation cartridge. [Figure 4B] This image shows a zoomed-in view of the lower region of a cylindrical sample preparation cartridge according to one embodiment of the present disclosure. [Figure 4C] This image shows a further zoomed-in view of the lower region of a cylindrical sample preparation cartridge according to one embodiment of the present disclosure. [Figure 4D] An embodiment of the present disclosure shows a cylindrical sample preparation cartridge. [Figure 4E] The sample preparation cartridge is shown with the film forming the side walls of the chamber removed to reveal the channels located on the chamber and annular wall. [Figure 4F] The sample preparation cartridge is shown with the film forming the side walls of the chamber removed to reveal the channels located on the chamber and annular wall. [Figure 4G] The chamber 103 is shown having a shelf baffle 108 that extends traversing through the chamber. [Figure 4H] The image shows a chamber 103 having a bottom wall that rises at one end. [Figure 5] A sample preparation system including a sample preparation cartridge 100 and a cylinder housing 130 according to an embodiment of the present disclosure is shown. [Figure 6] A diagram of an interfacial boundary between an air phase in an intermediate chamber and aqueous phases in two chambers adjacent to the intermediate chamber is shown. [Figure 7] Results obtained using air or oil as immiscible phases are shown. **DETAILED DESCRIPTION**

[0009] Aspects of the present disclosure include a sample preparation method, a sample preparation cartridge, and a sample preparation system.

[0010] The method for sample preparation disclosed herein utilizes an air phase to reduce the aqueous phase associated with magnetic particles bound to nucleic acids and to reduce carry-over of one or more contaminants such as cell debris, chaotropic agents, non-specifically attached molecules, and the like. This air transfer step is improved by using a combination of a first population of magnetic particles and a second population of magnetic particles, wherein the first population of magnetic particles is capable of associating with nucleic acids and the magnetic particles in the second population are at least twice as large in size as the magnetic particles in the first population. As discussed herein, the use of this second population of magnetic particles improves the transfer of the first population of magnetic particles by reducing the loss of the first magnetic particles during transfer. The sample preparation method can be semi-automatic or fully automated.

[0011] Before the sample preparation cartridge and method are described in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described and, of course, can vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0012] When a range of values is provided, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of the range, to one tenth of the unit of the lower limit, and any other stated value or intermediate value within the stated range of this description, is understood to be included in the present sample preparation cartridge, method, and sample preparation unit. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are likewise included in the sample preparation cartridge, method, and sample preparation unit, in accordance with any specifically excluded limits within the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are likewise included in the sample preparation cartridge, method, and sample preparation unit.

[0013] Certain ranges having numerical values preceded by the term "about" are presented herein. As used herein, the term "about" is used to provide literal support for the exact number preceding it, and for numbers that are close to, or approximate, the number preceding the term. When determining whether a number is close to, or approximate to, a specifically listed number, the close or approximate unlisted number can be a number that provides a substantial equivalent of the specifically listed number in the context in which it is presented.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present sample preparation cartridge, method, and sample preparation unit, but representative and exemplary sample preparation cartridges, methods, and sample preparation units are described below.

[0015] All publications and patents cited herein are incorporated herein by reference, and by being incorporated herein by reference, disclose and describe the relevant methods and / or materials from which those publications are cited, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Any citation of a publication is of its disclosure prior to the filing date, and the present invention should not be construed as acknowledging that such publication has no prior rights by features of the prior invention. Furthermore, the dates of the publications provided may differ from the actual publication dates, and these may need to be verified independently.

[0016] It should be noted that, as used herein and in the appended claims, the articles "a," "an," and "the" refer to multiple subjects unless otherwise explicitly indicated by the context. It should also be noted that claims may be designed to exclude any optional elements. Therefore, this statement is intended to function as an antecedent for the use of exclusive terms such as "solely" and "only," or for the use of "negative" limitation, in relation to the enumeration of claim elements.

[0017] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the sample preparation cartridge, method and sample preparation unit. Any enumerated method may be performed in the order of the enumerated events or in any other logically possible order.

[0018] Method for sample preparation As summarized above, aspects of this disclosure include methods for sample preparation. The terms “sample preparation” and “sample processing” are used interchangeably herein and refer to the process for isolating a target analyte, for example, cells, viruses, proteins, or nucleic acids present in a sample. This process involves binding the target analyte present in an aqueous phase to magnetic particles capable of binding to the analyte, an air transfer step to reduce the aqueous phase associated with the magnetic particles, and an elution step to release the target analyte bound to the magnetic particles. As will be discussed in detail elsewhere, the sample may be pre-treated before binding to the magnetic particles. If the sample does not contain a free target analyte, for example, a protein or nucleic acid, i.e., if the protein or nucleic acid is present in a microorganism or cell, the sample preparation methods disclosed herein may involve sample dissolution to release the protein or nucleic acid into an aqueous solution.

[0019] According to a particular embodiment, a method for processing a sample may involve contacting the sample with a first and a second collection of magnetic particles in an aqueous phase in a first region of a sample preparation cartridge. The first collection of magnetic particles is capable of associating with a target analyte, and the magnetic particles in the second collection are at least twice as large in size as the magnetic particles in the first collection. The method further includes transporting the first and second collections of magnetic particles from the aqueous phase in the first region to the air phase in the second region of the cartridge by applying a magnetic force to the magnetic particles. As used herein in the context of magnetic particles and analytes, the term “associable” means that the magnetic particles can bind to the analyte. Magnetic particles can be functionalized using standard methods so that they can bind to a target analyte. The functionalization is sufficiently specific under conditions such as the presence of a lysis buffer. For example, magnetic particles may be functionalized to bind to nucleic acids or proteins. In a particular example, nucleic acids may be attached to the surface of PMP by silica or iron oxide nucleic acid chemistry.

[0020] The magnetic particles in the first group may have a diameter of 500 nm to 10 μm. For example, the average diameter of the particles in the first group may be approximately 500 nm to 3 μm, 2 μm to 6 μm, 4 μm to 7 μm, or 8 μm to 10 μm. The magnetic particles in the second group may have a diameter that is 2 to 20 times the diameter of the magnetic particles in the first group, for example, 2 to 10 times, 3 to 10 times, or 3 to 5 times the diameter of the magnetic particles in the first group. For example, the average diameter of the particles in the first group may be 1 μm to 3 μm, and the average diameter of the particles in the second group may be 6 to 60 μm, for example, 9 μm to 50 μm, 10 μm to 30 μm, or 10 μm to 20 μm. In other examples, the average diameter of the particles in the first group may be 2um to 6um, and the average diameter of the particles in the second group may be 10 to 60um, for example 10um to 50um, 10um to 30um, or 10um to 20um. In other examples, the average diameter of the particles in the first group may be 3um to 7um, and the average diameter of the particles in the second group may be 10 to 140um, for example 10um to 120um, 20um to 120um, or 50um to 120um. In yet another example, the average diameter of the particles in the first group may be 8um to 10um, and the average diameter of the particles in the second group may be 20 to 60um, for example 20um to 50um, or 20um to 30um.

[0021] The ratio of the amounts of the first and second magnetic particles may be 1:1, 2:1, 1:2, 3:1, 1:3, 1:10, 1:30, 1:100, etc. In certain cases, the ratio of the amounts of the first and second magnetic particles may be 1:1. As used herein, amount refers to the mass of magnetic particles. The amount of magnetic beads in the second group may vary. It may be as low as 1% or 99% of the total bead mass per reaction.

[0022] As used herein, magnetic particles refer to particles that respond to magnetism. Magnetically responsive particles include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Suitable examples of paramagnetic materials include iron, nickel, and cobalt, as well as Fe3O4, BaFe 12 O 19 Examples include metal oxides such as CoO, NiO, Mn2O3, Cr2O3, and CoMnP. The first and second magnetic particles may each contain the same magnetically responsive material. For example, both types of particles may be made from the same paramagnetic material, e.g., iron oxide, e.g., ferromagnetic and / or ferrimagnetic material, cobalt oxide, nickel oxide, and mixtures thereof. In certain embodiments, the first and second magnetic particles do not contain a significant amount of non-magnetic elements, i.e., elements that are not polarized in a magnetic field and therefore not attracted to a magnet. Such non-magnetic elements include second silver, gold, copper, etc. In certain embodiments, the first and second groups of magnetic particles include paramagnetic materials surrounded by a non-magnetic polymer, e.g., magnetic materials covered with a polymer material, or magnetic materials embedded in a polymer matrix. Such particles may be referred to as magnetic beads or paramagnetic beads. The magnetic particles of a second group of magnetic particles (also referred to as "second magnetic particles") may contain more paramagnetic material and have a larger diameter compared to the magnetic particles of the first group of magnetic particles (also referred to as "first magnetic particles"). As a result of the size difference, the second magnetic particles experience a stronger attraction from the magnetic field compared to the first magnetic particles. However, due to their proximity to the first magnetic particles, the second magnetic particles can be pulled along the first magnetic particles, thus increasing the overall magnetic attraction on the first magnetic particles.

[0023] In certain embodiments, the second magnetic particles may be paramagnetic, while the first magnetic particles may be superparamagnetic. In other embodiments, the second magnetic particles may be superparamagnetic, while the first magnetic particles may be paramagnetic. In some examples, the first group of magnetic particles may include magnetic particles coated with silica. In some examples, the second group of magnetic particles may include magnetic particles coated with agarose, cepharose, or polystyrene. The magnetic particles may be magnetic microparticles and nanoparticles, and / or superparamagnetic microparticles and nanoparticles.

[0024] The sizes of the first and second magnetic particles are described in terms of diameter, but the particles do not necessarily have to be spherical in size and may be amorphous. In the case of amorphous particles, the diameter is the maximum distance from one side to the opposite side in the diametrical direction. In certain embodiments, the magnetic particles may be substantially spherical. Exemplary magnetic particles are those available from commercial sources such as Dynabeads® magnetic beads provided by Invitrogen, Estapor® superparamagnetic microspheres and PureProteome® magnetic beads by Merck Millipore, BcMag® by Bioclone Inc., ProMag® and BioMag® from Bangslabs, SupraMag® by Polymicrospheres Inc., TurboBeads® by Turbobeads Llc., and SPHERO® polystyrene magnetic particles by Spherotech. Superparamagnetic beads are available from Sigma-Aldrich and Thermo Scientific.

[0025] The first and second magnetic particles may be functionalized to bind to target analytes, such as nucleic acids like DNA and RNA. In certain embodiments, only the first magnetic particles are functionalized to bind to the target analyte, while the second magnetic particles do not bind to the target analyte to a noticeable extent. In some embodiments, using smaller diameter magnetic particles functionalized to bind to the target analyte reduces nonspecific binding by other molecules present in the sample.

[0026] According to a particular embodiment, the method may further involve transporting first and second groups of magnetic particles from the air phase in a second region to the aqueous phase in a third region of the cartridge by applying a magnetic force to the magnetic particles.

[0027] In a particular example, when a magnetic force is applied, magnetic particles aggregate in an area in a first region adjacent to the source of the magnetic force, and transport involves maintaining the magnetic force on the aggregated magnetic particles, moving the aggregated magnetic particles to the air phase in a second region of the cartridge, and moving the aggregated magnetic particles to the aqueous phase in a third region of the cartridge.

[0028] In some cases, transporting the first and second sets of magnetic particles involves moving a magnet that generates a magnetic force relative to different areas of the cartridge. In other cases, transporting the first and second sets of magnetic particles involves moving the cartridge or a portion thereof relative to a magnet that generates a magnetic force.

[0029] As will be described in more detail in the following section, the disclosed method does not depend on a specific configuration of the sample preparation cartridge. While an exemplary configuration of the sample preparation cartridge is described, other configurations may also be used.

[0030] In certain examples, the sample preparation cartridge may be a planar sample preparation cartridge. For example, the cartridge may be rectangular or circular but have a low profile so as to be substantially flat. In another example, the cartridge may be substantially cylindrical. In yet another example, the cartridge may include a first region which is a chamber and a separable second region which is a plate, and this cartridge is used in conjunction with another cartridge which includes a third region through which magnetic particles are transported.

[0031] In one example, the cartridge is substantially planar and comprises a first plate positioned spaced apart from a second plate, with the first and second plates held in a stationary position relative to each other. In a particular embodiment, the cartridge is substantially planar and comprises a first plate positioned spaced apart from a second plate, with the first and second plates movable relative to each other such that the plates are held spaced apart and slidable. The cartridge may be substantially planar and may not include a separate chamber, the aqueous phase in the first region may be aqueous droplets, the air phase in the second region is air present between the first and second plates, and the aqueous phase in the third region, if present, is aqueous droplets. Sample preparation devices having one or more of the features of such cartridges are described in U.S. Patent No. 9,766,166, which is incorporated herein by reference. See, for example, Figures 1A-1G of U.S. Patent No. 9,766,166.

[0032] In a particular embodiment, the first region is a first chamber containing an aqueous phase, and the second region is a second chamber containing an air phase. The first and second chambers are connected via a first channel, and the pressure difference between the first and second chambers establishes a liquid-air interface within the first channel. In a particular example, the cartridge includes a third region, which is a third chamber containing an aqueous phase.

[0033] In another embodiment, the disclosed method includes transporting first and second collections of magnetic particles from an air phase in a second region to an aqueous phase in a separate sample preparation cartridge, wherein transport includes maintaining a magnetic force on the magnetic particles until the second region is connected to the aqueous phase in the separate cartridge, and then removing the magnetic force, thereby allowing the magnetic particles to be released into the aqueous phase. The first and second regions of the cartridge may be detachably connected. For example, the first region is a first chamber, the second region is a transfer plate, and the third region is a third chamber. Sample preparation devices having one or more of the features of such a cartridge are described in U.S. Patent No. 10,040,062, which is incorporated herein by reference. See, for example, Figures 1-5 of U.S. Patent No. 10,040,062.

[0034] According to a particular embodiment, a method for sample processing involves bringing a sample into contact with a first group of magnetic particles and a second group of magnetic particles in a first chamber of a sample processing cartridge, wherein the first group of magnetic particles is capable of associating with a target analyte, such as nucleic acids, and the magnetic particles in the second group have a diameter at least twice as large as the diameter of the first group of magnetic particles, and transporting the first and second groups of magnetic particles from the first chamber to the second chamber of the cartridge by contacting and applying a magnetic force to the particles, wherein the second chamber contains air, and The invention may include transporting first and second groups of magnetic particles from the second chamber to a third chamber containing an aqueous solution, wherein the first and second chambers are connected via a first channel, and the pressure difference between the first and second chambers establishes a liquid-air interface in the first channel, and the transporting of first and second groups of magnetic particles from the second chamber to a third chamber containing an aqueous solution by applying a magnetic force to the particles, wherein the second and third chambers are connected via a second channel, and the pressure difference between the second and third chambers establishes an air-liquid interface in the second channel, and the transporting of magnetic particles. Exemplary cartridges that can be used in such a manner are further described in the following sections, schematically illustrated in Figures 1A-1B, and shown in Figures 1C-1D.

[0035] Contact can be carried out under conditions sufficient to bind the target analyte (e.g., nucleic acid) present in the sample to at least a first population of magnetic particles. In certain embodiments, the first chamber of the sample processing cartridge may contain an aqueous phase in which magnetic particles are present and capable of binding to the target analyte. In certain embodiments, the aqueous phase may be a lysis buffer. The lysis buffer may be a standard lysis buffer as known in the art. For example, the lysis buffer may contain a chaotropic agent that causes lysis of microorganisms such as bacteria and viruses, as well as cells such as mammalian cells. In certain examples, guanidine hydrochloride may be used as the chaotropic agent.

[0036] The contact step may optionally involve stirring the aqueous phase containing the sample mixture and the first and second populations of magnetic particles, and optionally the aqueous phase may contain a lysis buffer. The contact may be carried out for a period of time sufficient for the nucleic acid to bind to at least the first population of magnetic particles.

[0037] In some embodiments, contacting a sample with first and second groups of magnetic particles includes contacting the sample with an aqueous solution containing the first and second groups of magnetic particles. In some embodiments, contact includes placing the sample in a first chamber and subsequently introducing the first and second groups of magnetic particles and the aqueous solution into the first chamber. In some embodiments, the first chamber contains the first and second groups of magnetic particles, and introducing the aqueous solution moistens the magnetic particles and causes them to disperse. In some embodiments, the first chamber comprises a compartment containing the first and second groups of magnetic particles, and introducing the aqueous solution moistens the magnetic particles and causes them to flow from the compartment into the first chamber. The aqueous solution may be a lysis buffer. The lysis buffer does not contain free nucleic acids and can be used to process samples containing nucleic acids present in cells or viruses.

[0038] Many types of samples can be processed using the methods, cartridges, and systems of this disclosure. Samples contain, or are suspected of containing, cells, viruses, proteins, or nucleic acids. In certain examples, the material of interest may be nucleic acids present within cells or viruses. In certain embodiments, contact results in the destruction of cells or viruses present in the sample, causing them to release nucleic acids present within the cells or viruses, respectively.

[0039] In certain embodiments, contact involves stirring a mixture containing the sample and first and second collections of magnetic particles. Stirring may be used to facilitate cell / virus lysis and / or to ensure uniform dispersion of the magnetic particles. In certain embodiments, stirring may involve shaking the cartridge. Shaking may be achieved using a rotary shaker or vortexer. In certain embodiments, the sample preparation cartridge may be cylindrical in shape and may rotate by reciprocating around a central axis extending between the upper and lower ends of a cylinder.

[0040] In certain embodiments, the method involves transporting first and second collections of magnetic particles from a first chamber to a second chamber of a cartridge by applying a magnetic force to the particles. The second chamber of the sample processing cartridge may be filled with air, for example, compressed air. Compressed air can be generated by filling the first and third chambers with aqueous solutions at atmospheric pressure. For example, at the start of the method, all three chambers may be empty and therefore contain only air. During sample processing, the first and third chambers are filled with an aqueous phase. The aqueous phase pushes the air present in the first and third chambers into the second (intermediate) chamber, resulting in compression of the air present in the second chamber. The aqueous solution also flows into and partially fills a first channel extending between the first and second chambers, and a second channel extending between the second and third chambers. Interfaces formed in the first and second channels between the aqueous and gas phases act as barriers preventing aqueous substances from leaving the aqueous phase and entering the gas phase. Therefore, the interface reduces the carryover of aqueous solution trapped on or between the magnetic beads from the first chamber to the second chamber.

[0041] The method further includes transporting first and second groups of magnetic particles from a second chamber to a third chamber of a cartridge by applying a magnetic force to the particles. In certain embodiments, the third chamber contains an aqueous phase which may be a washing buffer or an elution buffer. In certain embodiments, the third chamber contains an elution buffer. In certain embodiments, the third chamber contains a washing solution, and the cartridge comprises a fourth chamber containing air or an immiscible substance and a fifth chamber containing an elution buffer. The sample processing method further includes transporting first and second groups of magnetic particles from the third chamber through the fourth chamber to the fifth chamber.

[0042] In some embodiments, transporting first and second collections of magnetic particles from one chamber to adjacent chambers of a cartridge by applying a magnetic force to the particles involves placing a magnet adjacent to the chamber to induce the formation of aggregates containing the magnetic particles, the magnet positioned such that the aggregates are spatially aligned with the entrances to the first and second channels.

[0043] Transporting the first and second collections of magnetic particles may include moving a magnetic field relative to the cartridge while the cartridge remains stationary, moving the cartridge relative to the stationary magnetic field, and / or moving the device cartridge and the magnetic field. In certain embodiments, the method may involve, after the contact step, exposing the magnetic particles to a magnet to aggregate them, and using the magnet to transport the aggregated magnetic particles. In certain embodiments, the method involves applying a magnetic force to the magnetic particles to form aggregates of the first and second collections of magnetic particles, the aggregates being spatially aligned with the entrance to the first channel. In other words, the magnet is positioned relative to the sample preparation cartridge such that the magnetic particles move to an area in the cartridge adjacent to the magnet, which is spatially aligned with the entrance to the first channel. Generally, the areas where the magnetic particles aggregate are the inner surfaces of the walls of the sample preparation cartridge, e.g., the walls forming one of the first, second, and third chambers and the sides of the first and second channels. In a particular embodiment, the first chamber of the sample preparation cartridge has a side that decreases in size from the first chamber to the first channel, facilitating the transport of aggregated particles from the first chamber to the second chamber through the first channel. This tapered inlet to the first channel can facilitate not only the transport of compactly aggregated magnetic particles adjacent to the magnet, but also the transport of loosely aggregated magnetic particles, which may lag behind the magnetic force moving the compactly aggregated magnetic particles.

[0044] As mentioned above, the arrangement of the magnets may be such that the aggregate is spatially aligned with the entrance to the second channel, and therefore it is not necessary to further move the aggregated magnetic particles to move them to the entrance. In other cases, the aggregated magnetic particles may be moved by magnetic force to align the aggregate with the entrance to the first channel.

[0045] In a particular embodiment, the inlet to the second channel includes a tapered region that decreases in size from the second chamber to the second channel, facilitating the transport of aggregates from the second chamber to the third chamber through the second channel.

[0046] In some embodiments, transporting first and second collections of magnetic particles from a first chamber to a second chamber of a cartridge by applying a magnetic force to the particles involves placing a magnet adjacent to the first chamber to induce the formation of aggregates containing the magnetic particles, the magnet positioned such that the aggregates are spatially aligned with the entrances to the first and second channels.

[0047] According to a particular embodiment, the outer magnet may be positioned at a distance of 1 cm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less, or 0.5 mm or less, or 0.2 mm or less, or 0.1 mm or less from the outer surface of the cartridge forming the wall of the chamber / region where the magnetic particles are present. In a particular example, the wall of the chamber / region where the magnetic particles are present may have a thickness of less than 2 mm, less than 1 mm, or less than 0.5 mm, for example, 0.1 mm to 5 mm, or 0.2 mm to 4 mm, or 0.5 mm to 2 mm.

[0048] In some embodiments, the method is semi-automatic. For example, at least one step of the method may be performed by an instrument instead of a user. In a particular embodiment, the step of bringing the sample into contact with a first and second collection of magnetic particles in a first chamber of a sample preparation cartridge includes loading the sample into the first chamber of the sample preparation cartridge by the user, and one or more of the remaining steps are performed automatically. In some cases, the step of stirring the mixture of the sample and the first and second collections of magnetic particles may be performed automatically. In some cases, the step of transporting the magnetic particles may be automated. Automation may be achieved, for example, by a computer that controls the movement of the sample preparation cartridge and / or magnets.

[0049] In certain embodiments, the method may further include agglomerating magnetic particles in a third chamber (or fifth chamber) filled with an aqueous solution, such as an elution buffer. This chamber may also be referred to as an elution chamber. Once the magnetic particles are agglomerated and their movement is prevented, an aqueous solution containing nucleic acids isolated from the sample (e.g., an elution buffer such as PCR amplification buffer) may be withdrawn. The aqueous solution may be withdrawn by pipetting out manually or automatically. The aqueous solution may also be withdrawn by draining it from the third (or fifth) chamber, for example, by forcing it through a hole. The elution chamber may be connected to a collection container from which the aqueous solution can be drained.

[0050] An exemplary method is schematically illustrated in Figures 2 and 3. Figure 2 shows a sample preparation method using a sample processing cartridge having three chambers. The method includes mixing the sample with a lysis buffer or solvent and paramagnetic particles (PMPs) so that the PMPs are in solution (Step 1). The PMPs are agglomerated by bringing an outer magnet close to the cartridge, and the agglomerated PMPs are transported from the aqueous phase containing the lysis buffer to the air phase (Step 2). In Step 3, the agglomerated PMPs are transported from the air phase to the chamber containing the elution buffer.

[0051] Figure 3 shows a sample preparation method using a sample processing cartridge with five chambers. The method involves mixing the sample with a lysis buffer and paramagnetic particles (PMPs) so that the PMPs are in solution (Step 1). The PMPs are agglomerated by bringing an outer magnet close to the cartridge, and the agglomerated PMPs are transported from the aqueous phase containing the lysis buffer to the air phase, and then transported to a third chamber containing a washing buffer (e.g., alcohol-methanol or ethanol) (Step 2). In Step 3, the agglomerated PMPs are resuspended by removing the magnet and optionally agitating the cartridge. The PMPs are then captured again using the magnet and transported to a chamber containing an elution buffer.

[0052] The samples processed by the methods, cartridges, and systems provided herein may be biological samples, for example, the sample may be a sample of whole blood, serum, plasma, sputum, nasal fluid, saliva, mucus, semen, vaginal fluid, tissue, urine, organs, and / or similar of a mammal (e.g., human, rodent (e.g., mouse), or any other mammal of the subject). In other embodiments, the sample may be a collection of cells from a non-mammalian source, such as bacteria, yeast, insects (e.g., fruit flies), amphibians (e.g., frogs (e.g., xenopus)), viruses, plants, or any other non-mammalian nucleic acid sample source.

[0053] Sample preparation cartridge A sample preparation cartridge used to carry out the methods disclosed herein may include a plurality of sample processing regions or chambers, each containing reagents for the purification, modification, analysis, and / or detection of biomolecules or cells, and a gas phase (air) between (e.g., separating) two or more of the regions / chambers. In some embodiments, there are three chambers. In some embodiments, there are four chambers. In some embodiments, there are five chambers. In some embodiments, there are six or more chambers (e.g., seven, eight, nine, ten, eleven, and up to thirtieth chambers). In some embodiments, the air provides a continuous barrier between two or more of the chambers (i.e., the sample passes from the aqueous phase into the air and out of the air directly into the next aqueous phase).

[0054] Exemplary devices that may be used to prepare a sample according to the method of this disclosure are depicted in Figures 1A–1G of U.S. Patent No. 9,766,166, Figures 1–5 of U.S. Patent No. 10,040,062, and Figure 2 of U.S. Patent No. 8,304,188. The disclosure of these devices is incorporated herein by reference. Other devices that may be used to carry out the method for sample processing are schematically depicted in Figures 1A–1B and shown in Figures 1C–1D.

[0055] In certain examples, the method may be carried out using a sample preparation cartridge. The sample preparation cartridge may have a substantially cylindrical shape. For example, the sample preparation cartridge includes a cylindrical structure including an upper end, a lower end, and an annular wall extending between the upper and lower ends. The cylindrical structure includes a plurality of chambers arranged in the annular wall, each chamber extending between the outer surface of the annular wall and the inside of the cylindrical structure, the annular wall having cavities that form the opening side of each chamber; one or more channels providing fluid communication between the plurality of chambers, formed by recesses in the annular wall and having opening sides; and one or more covers attached across the outer surface of the annular wall to cover and fluidly seal the opening sides of the chambers and the opening sides of the recesses.

[0056] In addition, in certain embodiments, the sample preparation cartridge may also include a buffer pack, a sealing lid assembly, a protective cover, and a cap. The cartridge may also include a sample loading component. The sample preparation cartridge is configured for use with a cylinder housing equipped with a magnet.

[0057] The term "cylindrical" means that the cylindrical structure can be substantially a right cylinder. The cylindrical structure may be rotatable around an axis formed by a line connecting the center of the bottom end of the cylindrical structure and the center of the top end of the cylindrical structure. For example, the cylindrical structure may rotate clockwise or counterclockwise when viewed from above, looking down at the top of the cylindrical structure. Alternatively, the cylindrical structure may rotate both clockwise and counterclockwise. The rotation of the cylindrical structure may be used to mix the contents of one or more chambers, or to position magnets located in cylinder housings adjacent to chambers to cause aggregation of magnetic particles present in the chambers, and / or to transfer the aggregated magnetic beads from one chamber to another.

[0058] As summarized above, the cylindrical structure comprises multiple cavities within the annular wall that form a chamber with multiple sides open on the annular wall. For example, the multiple cavities may be recesses within the annular wall that deform the continuous surface of the annular wall. "Sides open" means that the annular wall does not cover such sides of the chamber. In certain cases, the deformed annular wall may form closed sides of the chamber, and areas corresponding to the sides of the annular wall deformed to form cavities may form open sides of the chamber.

[0059] According to a particular embodiment, the opening sides of multiple chambers are located outside the annular wall. For example, the annular wall may be deformed inward from the outside to form an inwardly deformed cavity within the annular wall. In such a case, the opening sides of the chambers may be areas corresponding to the sides of the annular wall that have been inwardly deformed to form the cavity. In such cases, the inwardly deformed annular wall may form the closed sides of the chambers. The volume of the chamber may represent a measurement corresponding to the volume of the recess within the annular wall. The chamber may be any convenient volume, and in some cases, 1 cm 3 ~3cm 3 or 2cm 3 ~5cm 3 etc., 1cm 3 ~about 5cm 3 It may vary up to a certain point. In other cases, the chamber may contain a fluid of any convenient volume, which in some cases may vary from 1 μL to approximately 5,000 μL, such as 1 μL to 100 μL, or 1,000 μL to 3,000 μL, or 2,000 μL to 5,000 μL. Each chamber in a plurality of chambers may have the same volume or different volumes. The depth of the chamber, measured as the distance from the outer surface of the annular wall to the inner side of the chamber, may be of any convenient size, which in some cases may be 0.1 cm or more, such as 1 cm or 5 cm. Each chamber in a plurality of chambers may have the same depth or different depths.

[0060] According to a particular embodiment, multiple chambers are arranged in close proximity to each other on an annular wall. For example, the distance between the lateral boundary of a first chamber and the nearest lateral boundary of a second chamber may be about 0.1 cm or more, such as 0.5 cm to 1 cm, for example, 0.5 cm, 0.75 cm, or 5 cm. The distance between pairs of sides of adjacent chambers may be the same or different for multiple chambers.

[0061] As summarized above, the sample preparation cartridge includes one or more channels that provide fluid communication between a plurality of chambers. In certain embodiments, the channels are wide enough for one or more PMPs to be transported through them. In certain embodiments, one or more of the channels between chambers are formed by recesses in the annular wall. Recesses in the annular wall mean recesses or cavities in the annular wall that are capable of providing fluid communication between chambers. In some cases, the recesses are formed within the outer surface of the annular wall, thereby interconnecting a first chamber and a second chamber formed with open sides on the outer surface of the annular wall between such a first chamber and a second chamber by recesses in the outer surface of the annular wall. Recesses in the annular wall may have any convenient length, width, and depth. In certain examples, the height of the recess may be in the range of 0.5 mm to 5 mm, e.g., 2.5 mm; the depth may be 0.2 mm to 1 mm, e.g., 0.5 mm; and the length may be 1 mm to 10 cm, e.g., 4 to 5 cm.

[0062] In certain embodiments, the recesses are located on the sides of multiple chambers. The sides of multiple chambers refer to the left or right side of the chamber, rather than the upper or bottom side, when the axis of the cylindrical structure is oriented vertically, formed between the center of the bottom and the center of the top of the cylindrical structure. Placing the recesses on the sides of multiple chambers means that the recesses can interconnect the right side of a first chamber with the left side of a second chamber, thereby enabling fluid communication between such first and second chambers via the recesses. The recesses between chambers may be substantially straight lines between a point on the second chamber and a point on the first chamber opposite to it, so that the recess is substantially parallel to the plane defined by the bottom of the cylindrical structure. The recesses between the first and second chambers may have substantially the same width and depth in the annular wall along the entire length of the recess, or they may vary. Recesses between different pairs of chambers may have different or the same dimensions. The recesses may be conveniently shaped to allow PMP to be translated through them.

[0063] In certain embodiments, the recesses are located on the sides of one or more chambers at a substantially constant height above the bottom of the cylindrical structure. In these embodiments, the recesses between pairs of chambers may be substantially linear. In these embodiments, the recesses and chambers may be molded such that a linear path exists, starting from the left end of the leftmost chamber and passing through each of the multiple chambers to the rightmost chamber. The recesses on the sides of one or more chambers may be located at any convenient height above the bottom of the cylindrical structure. In certain embodiments of these embodiments, the height above the bottom of the cylindrical structure where the recesses are located corresponds to the vertical midpoint of one or more chambers.

[0064] In certain embodiments, the shape of one or more of the chambers is generally rectangular. A generally rectangular chamber means that the two-dimensional shape of the recess into the annular wall is longer in length than in width. The height and width of each chamber may be any convenient height and width. The height and width of each rectangular chamber may be the same or different.

[0065] In certain embodiments, the shape of a chamber connected to another chamber by one or more channels is such that, with respect to the lateral portion of the chamber adjacent to the channels, the height of the chamber at each lateral position of the chamber decreases as such position approaches the channels. In some cases, the height of such a chamber at each lateral position decreases linearly to form a tapered region. Such a tapered inlet into a recess can facilitate the transport of aggregated PMP from the chamber to the channels.

[0066] In certain embodiments, one or more of the chambers are provided with drainage holes. Drainage holes refer to openings through which fluid can pass and exit the chamber. For example, fluid may be discharged under the influence of gravity through drainage holes located at the bottom of the chamber. Alternatively, fluid may be ejected from the chamber by pressurizing the fluid within the chamber using a plunger.

[0067] One or more of the chambers may include openings configured for ventilation of the chamber, filling the chamber with fluid, and / or discharge of fluid from the chamber.

[0068] In certain embodiments, the inside of a cylindrical structure comprises one or more wells. A well means one or more enclosures within the interior of the cylindrical structure. The enclosures may be of any convenient size or shape. For example, an enclosure may be substantially cylindrical, having a closed bottom end, annular walls, and an open top end. In these embodiments, the cylindrical structure may further comprise channels within the cylindrical structure that provide fluid communication between such wells and one or more of the chambers. In some cases, each well is interconnected with a different chamber via one or more channels.

[0069] In certain embodiments, the multiple chambers form a first chamber, a second chamber, and a third chamber. In certain embodiments, the first chamber is adjacent to the second chamber, the second chamber is adjacent to the first and third chambers, and the third chamber is adjacent to the second chamber. In certain embodiments, the cylindrical structure further includes a first recess in the annular wall that provides fluid communication between the first chamber and the second chamber, and a second recess in the annular wall that provides fluid communication between the second chamber and the third chamber. In certain embodiments, the first chamber is a dissolution chamber, the second chamber is an immiscible phase chamber, i.e., air, and the third chamber is an elution chamber. A dissolution chamber means a chamber that contains a dissolution buffer, such as a fluid which is a dissolution buffer, during use of the sample preparation cartridge. An immiscible phase chamber means a chamber that contains an immiscible phase, such as a fluid which is immiscible with the aqueous phase, during use of the sample preparation cartridge. In some cases, the immiscibility phase may be, for example, oil, in which case the PMP is transported from the air chamber to the washing solution, then to the immiscibility phase chamber (oil or air), and finally to the elution chamber. The elution chamber refers to a chamber containing a fluid from which the analyte bound to the PMP can be released during use of the sample preparation cartridge. In certain embodiments, the fluid may be called an elution buffer. In certain embodiments, the elution buffer may be adapted for subsequent downstream processing of the isolated analyte. For example, the elution buffer may be an amplification buffer. The amplification buffer may be suitable for carrying out amplification of the isolated analyte, for example, by isothermal amplification or PCR.

[0070] The first chamber may include an opening at the top of the chamber. This opening may be configured as an inlet. The inlet may be configured to introduce a lysis buffer or solvent, a sample, and / or mixtures thereof. Therefore, the inlet may have a diameter suitable for pipetting, injecting, or pumping the lysis buffer, sample, and / or mixtures thereof. In some cases, the second chamber may also include an opening at the top of the chamber. This opening may be configured as an inlet for introducing an immiscible phase, such as oil, into the second chamber. In some cases, the third chamber may also include an opening at the top of the chamber. This opening may be configured as an inlet for introducing an elution buffer into the third chamber.

[0071] In certain embodiments, the first chamber may include a compartment located on or below the bottom region of the first chamber. The compartment may include an opening that fluidly connects the compartment to the inside of the first chamber. The compartment may include a first collection of magnetic particles and a second collection of magnetic particles, as described in the section on methods for sample preparation. The magnetic particles may be mixed together and then dried, for example, lyophilized. In certain embodiments, the magnetic particles may be mixed together, placed in a compartment, and subsequently dried to provide a lyophilized preparation. In certain embodiments, the magnetic particles may be mixed together, dried, and subsequently placed in a compartment to provide a lyophilized preparation. In certain embodiments, the first chamber includes an opening at the bottom of the chamber, which is configured as an inlet for a lysis buffer, and the first chamber has an opening at the top of the first chamber, which is configured as a sample inlet. In a particular embodiment, the compartment includes an inlet that fluidly connects the compartment to a channel and an outlet that fluidly connects the compartment to the inside of a first chamber.

[0072] In certain examples, the second chamber may not include any openings other than those connecting to the first and third chambers. The second chamber may contain air. When the first and third chambers are filled with liquid, the air in the second chamber is compressed due to the absence of vents in the second chamber. As described herein, the compressed air serves as a “cleaning” environment for the PMP, which is transferred from the first chamber through the second chamber containing the compressed air to the third chamber.

[0073] In certain examples, the third chamber includes an opening in the bottom region of the chamber. The opening is configured to fill the third chamber. The opening is distinct from a drain hole located in the bottom region of the chamber. In certain cases, the drain hole may have a smaller diameter than the opening configured to fill the third chamber, such that the drain hole does not allow liquid to pass through at atmospheric pressure and requires a higher pressure to allow liquid to pass through. In some cases, the drain hole at the bottom of the third chamber is fluidically connected to one or more collection containers. The collection containers may be two separate tubes, e.g., thin-walled polypropylene tubes suitable for PCR, or similar thin-walled containers or strips that facilitate a thermal circulation reaction. The drain hole at the bottom of the third chamber may branch off from the drain hole and be fluidically connected to two channels that fill the two collection containers with substantially equal volumes of liquid discharged from the third chamber.

[0074] Figure 4A shows a cylindrical cartridge according to one embodiment. In this example, the cylindrical cartridge 100 includes three cavities within the annular wall that form chambers 101, 102, and 103 with three sides open on the annular wall, and two recesses that form interconnects 104 with sides open. As can be seen, the open sides of chambers 101, 102, and 103 are located outside the annular wall, and chambers 101, 102, and 103 are located adjacent to each other. The two interconnects 104 between chambers 101, 102, and 103 provide fluid communication between the chambers. In this example, the interconnects 104 are channels which are recesses within the annular wall, and the interconnects 104 are located on the sides of the multiple chambers 101, 102, and 103. As shown in the figure, the recesses that form the interconnects 104 between chambers 101, 102, and 103 are at a substantially constant height above the bottom end of the cylindrical structure 100. Also shown in Figure 4A is a compartment 125 located in the bottom region of the first chamber. The compartment contains a dry mixture of first and second collections of magnetic particles disclosed herein. The compartment includes an opening 130 through which the magnetic particles can enter the first chamber. The compartment is fluidically connected to a channel connected to a buffer pack that supplies an aqueous phase, such as a lysis buffer, to the first chamber through the compartment. Figure 4B shows a magnified image of the cartridge for sample preparation, which shows the cartridge in an inverted orientation. The first chamber 101 is visible. The compartment 125 is also visible. The PMP 150 is located within the compartment 125. Figure 4C shows a photograph of the bottom region of the sample preparation cartridge viewed from below. The film covering the compartment 125 has been removed to help visualize the PMP 150. Also visible is a channel leading to the bottom region of the compartment 125, which can be used to supply an aqueous phase (e.g., lysis buffer) to the compartment.

[0075] A sample preparation cartridge includes one or more covers that close the opening sides of multiple chambers and interconnections that form channels. In certain embodiments, the covers are curved to fit with the outer surface of a cylindrical structure. When the covers close the chambers, the fluids disposed within the chambers are completely contained within them. The use of covers to form the chamber walls within a cylindrical cartridge allows for walls that are significantly thinner than the annular walls of the cylindrical structure. The use of covers to form the chamber walls within a cylindrical cartridge allows for walls made from materials different from those of the cylindrical structure.

[0076] The cover may be made of any suitable material that is curved and can be attached to the outer surface of the annular wall. For example, the cover may be made of plastic (e.g., thermoplastic resins such as cycloolefin polymers or cycloolefin copolymers), metal, paper, glass, etc. If a metal material is used for the cover, the metal may be nonmagnetic, i.e., it may not contain a significant amount of iron. Paper covers may include a non-wetting coating, e.g., a wax coating. The cover may be substantially opaque or substantially transparent. The cover may be attached to the annular wall by any suitable means, such as by adhesive, by locally heating the outside of the annular wall or the cover or both, or by screwing the cover into the annular wall by snapping the cover into a groove created in the annular wall. The cover may be thin enough not to significantly reduce the magnetic force of the outer magnet in the chamber. For example, the cover may be thin enough to allow paramagnetic particles (PMPs) present in the chamber to aggregate in response to an outer magnet positioned adjacent to the chamber, and to allow the aggregated PMPs to traverse channels connecting adjacent chambers in response to the relative movement of the cylindrical structure and the outer magnet. The cover may have a thickness of less than 1 cm, less than 0.5 cm, less than 0.1 cm, for example, 1 mm to 5 mm, or 0.1 mm to 5 mm, or 0.1 mm to 1 mm, or 0.1 mm to 0.5 mm. In certain embodiments, the cover may be a film, for example, an adhesive film.

[0077] According to a particular embodiment, the cover fluidly seals the open sides of multiple chambers. Fluidly sealing the open sides of multiple chambers means that, when the cover is placed on a cylindrical structure, the spaces within the chambers do not fluidly communicate with the spaces outside the cylindrical structure through the open sides of the chambers.

[0078] According to certain embodiments, the inner surface of the cover facilitates the movement of magnetic particles in contact with it. Facilitating the movement of PMPs means that the inner surface of the cover can be configured such that the PMPs can be more reliably translated from a first position on the cover to a second position on the cover while remaining in contact with the inner surface of the cover. For example, the inner surface of the cover may be polished to reduce friction between the PMPs and the inner surface of the cover as the PMPs move along the cover. Translation from a first position on the cover to a second position on the cover means, in certain cases, that the PMPs move along the inside of the cover.

[0079] In certain embodiments, the sample preparation cartridge may include a buffer pack. The buffer pack may include one or more fluid packs. Each fluid pack may contain a fluid. In some embodiments, the fluid pack may include a dissolution buffer pack and an elution buffer pack. In other embodiments, the fluid pack may include each of the dissolution buffer pack, an immiscible phase pack, and an elution buffer pack. In certain embodiments, the immiscible phase may contain oil. In certain embodiments, the immiscible phase may contain air. In some cases, one or more of the fluid packs may further contain PMP. The fluid pack may contain any convenient amount of PMP, measured, for example, based on the volume or weight of the PMP. For example, if the PMP is included in a fluid pack, it may be mixed with the fluid. In some cases, the PMP may be included in a fluid pack containing the dissolution buffer.

[0080] In certain embodiments, the buffer pack is configured to fit into a well of a cylindrical structure. For example, if the well is molded as a substantially hollow cylinder, the buffer pack may be molded as a cylinder that fits into the well of the cylindrical structure. The buffer pack is described in more detail in the U.S. Provisional Patent Application, “Magnetic Particle Separation Device Buffer Pack and Cap Design,” co-filed with this application, attorney reference number ADDV-082PRV, which is incorporated herein by reference in its entirety.

[0081] In some embodiments, the lysis buffer can be formulated to release nucleic acids from a wide range of samples, such as tissue samples, cells, viruses, or bodily fluid samples. The lysis buffer can also be designed to lyse all types of pathogens, such as viruses, bacteria, fungi, and protozoan pathogens. Such a lysis buffer may contain chaotropic agents, particularly guanidine hydrochloride. In addition, the lysis buffer may contain other reagents such as surfactants, defoamers, and buffers.

[0082] In a particular embodiment, the sample preparation cartridge includes a sealing lid assembly. The sealing lid assembly includes a sealing plate positioned at the upper end of a cylindrical structure and a protective cover positioned across the sealing plate. The protective cover surrounds the sealing plate and can be snapped onto and around the upper region of the cylindrical structure to hold the sealing plate in place.

[0083] The sealing plate fits onto the upper end of the cylindrical structure, closing the upper end. In certain embodiments, the sealing plate may include an opening adjacent to an opening in a third (elution) chamber for withdrawing elution buffer for analysis of eluted nucleic acids. In other embodiments, the sealing plate may further include a plunger assembly. The plunger assembly may include a gasket seal mounted on a shaft, a spring, and a trigger for engaging the spring with the shaft. The shaft may be of any convenient length, such as a length less than or equal to the height of the corresponding chamber. The gasket seal may be molded such that the size of the working end of the gasket seal is substantially the same as that of the corresponding chamber into which the plunger is integrated. In these embodiments, the spring may apply tension to the plunger in the retracted position; that is, when the plunger is retracted, the spring is under tension. Retraction means that the gasket seal end of the plunger is retracted. When in the retracted position, the plunger cannot eject fluid from the corresponding chamber. The amount of tension applied by the spring when the plunger is retracted corresponds to the amount of tension applied to the plunger by the spring when the plunger is no longer retracted, and can be varied as desired. The trigger that engages the spring with the shaft means that the trigger can control the release of the spring under the tension that holds the plunger in the retracted position.

[0084] In certain embodiments, the trigger and spring are mechanically interconnected such that the trigger is activatable when the plunger is in the retracted position. "Activatable" means that pressing the trigger releases the tension in the spring, thereby moving the plunger from the retracted position to the extended position.

[0085] In these embodiments, the plunger gasket seal may be positioned to engage with one of the chambers. Engaging with one of the chambers means that the plunger assembly is positioned such that when the plunger assembly is in the protruding position, the plunger gasket seal substantially fills the bottom portion of the chamber, and when the plunger assembly is in the retracted position, the plunger gasket seal does not fill the bottom portion of the chamber. That is, the movement of the plunger from the retracted position to the protruding position is such that the plunger can protrude from the chamber. Protruding from the chamber means that when the plunger transitions from the retracted position to the protruding position, the plunger gasket seal engages with the chamber and pressurizes any fluid within the chamber.

[0086] In these embodiments, the trigger may be positioned on the sealing lid assembly so as to protrude beyond the outer wall of the cylindrical structure. Protruding beyond the outer wall of the cylindrical structure means that the distance between the axis of the cylindrical structure and the furthest point on the trigger is greater than the distance between the axis of the cylindrical structure and the outer edge of the annular wall. The trigger can protrude at any convenient distance beyond the outer edge of the annular wall. In these embodiments, the trigger may be oriented to be pressed laterally. Pressing the trigger means activating the trigger to release the tension of a mechanically coupled spring. Oriented to be pressed laterally means that the trigger is positioned such that it must be moved substantially laterally in order to press it.

[0087] As summarized above, in certain embodiments, the sample preparation cartridge further includes a cap slidably positioned on top of a cylindrical structure. Slidably positioned means that the cap can be positioned on top of the cylindrical structure so that it can slide toward the cylindrical structure.

[0088] In certain embodiments, the cap may comprise one or more arms arranged to mechanically engage with the buffer pack. For example, the cap may have a substantially flat shape, with one or more arms attached to one flat side of the cap. Such arms may be of any convenient size or shape. For example, the length of the arms may be long enough to reach the wells inside the cylindrical structure when the cap is positioned on top of the cylindrical structure.

[0089] In certain embodiments, the cap may include a plunger, which, when the cap is slid within a cylindrical structure, is positioned to enter the sample chamber and discharge the sample into the dissolution chamber. The sample chamber may be adjacent to the first (dissolution) chamber and connected to the dissolution chamber via a channel. One of the arms of the cap may enter the dissolution buffer pack and force the dissolution buffer out of the pack into the first (dissolution) chamber. Another arm of the cap, if present, may enter the immiscibility phase pack and push the oil into the second (immiscibility phase) chamber, while a third arm of the cap may enter the elution buffer pack and push the elution buffer into the third (elution) chamber.

[0090] The cartridge may be loaded into an instrument equipped with a magnet, in which case the magnet is positioned relative to the cartridge so that it can be used to move the PMP from the dissolution chamber through the immiscibility phase chamber into the elution chamber. The instrument may include a motor that engages with the cartridge to rotate the cartridge relative to the magnet, or the magnet may be configured to move along the annular surface of the cartridge.

[0091] A sample preparation cartridge according to one embodiment of the present disclosure is shown in Figure 4D. In this example, the sample preparation cartridge 400 includes a cylindrical structure 410, a cover 420, and a cap 430.

[0092] Figures 4E-4G illustrate a sample preparation cartridge that includes certain modifications to the sample preparation cartridge shown in Figures 4A-4D. Figure 4E shows an air isolation path 105 located between chambers 101 and 102, and an air chamber 106 located between chambers 102 and 103. In this example, the air isolation path 105 is connected to the air gap chamber 106. In other examples, the air isolation path 105 may not be connected to the air gap chamber 106. For example, the air isolation path may terminate in the bottom region of the cartridge. In certain embodiments, the bottom region of the air gap chamber may be closed. The width of the air gap may be shorter than the width of the air chamber, or vice versa. In other examples, the widths of the air gap and the air chamber may be the same. In certain examples, a baffle 107 may be introduced into chambers 102 and / or 101. Figure 4F illustrates a chamber 102 having a baffle 107 that can prevent liquid from splashing into the channel 104 during mixing of magnetic beads by the reciprocating rotation of the cartridge, for example. The baffle 107 may be positioned below the channel 104, for example, 2mm to 10mm, 3mm to 8mm, or 5mm to 7mm below the channel 104. The baffle may have a maximum width of 5mm, for example, 1mm to 3mm, when measured from the side wall of the chamber on which the baffle extends.

[0093] Figure 4G shows chamber 103, with a shelf baffle 108 extending traversing through the chamber. The shelf baffle 108 includes a notch 109 and an opening 190 to allow magnetic beads to pass into the area below the shelf baffle. The shelf baffle may be used to prevent splashing of liquid during mixing of liquids present in chambers 102 and / or 103.

[0094] Figure 4H shows a modified chamber 103 having a bottom wall 111 that rises at one end such that the bottom wall closer to the channel 104 forms an acute angle with the side wall of the chamber 103. This configuration may be an alternative to using a shelf baffle to prevent liquid present in the chamber 103 from splashing into the channel 104.

[0095] Sample preparation system There is provided a sample preparation system comprising a sample processing cartridge described herein and a magnet operably arranged in association with the cartridge so as to apply a magnetic force to magnetic particles within the cartridge. An exemplary sample preparation system includes a cylinder housing in which a cylindrical sample preparation cartridge can be removably disposed. Removably disposed means that the cylindrical cartridge can be fitted into the cylinder housing such that the cylindrical cartridge can still be separated from the cylinder housing. For example, a user may dispose the cylindrical cartridge within the cylinder housing and may remove the cylindrical cartridge from the cylinder housing after sample preparation. As summarized above, the cylinder housing includes a magnet. A magnet means any object having the ability to generate a magnetic field outside itself. For example, a magnet can generate a magnetic field capable of attracting magnetic particles. In some instances, the magnet may be a permanent magnet or an electromagnet. As used herein, "magnet" refers to a material or article capable of spontaneously or actively generating a magnetic field, the strength of which can be measured by a conventional gaussmeter. The magnet can be a permanent magnet or an electromagnet. As used herein, "permanent magnet" refers to any object that is magnetized and creates its own permanent magnetic field. Suitable ferromagnetic materials for permanent magnets include iron, nickel, cobalt, rare earth metals, and their alloys. The term "permanent" does not mean that such a magnet cannot lose its magnetism, for example, by heat, physical shock, or exposure to an opposing magnetic field. In some examples, the permanent magnet is a samarium cobalt (SmCo) alloy, an aluminum nickel cobalt alloy (AlNiCo), a neodymium iron boron (NdFeB) alloy, Nd2Fe 14 B, or contains ferrite. As used herein, "electromagnet" refers to any device capable of creating a magnetic field through the application of electrical energy. An electromagnet can include a core and a coil or other elements for carrying an electric current to generate a magnetic field.

[0096] In certain embodiments, the magnet is positioned close to the outside of the annular wall of the cylindrical cartridge. In some embodiments, the magnet is located outside the cylindrical cartridge and is used to transfer magnetic particles between the chambers of the cylindrical cartridge.

[0097] In certain embodiments, the cylindrical cartridge rotates within the cylinder housing. Rotation means that the cylinder housing allows for degrees of freedom of the cylindrical cartridge to rotate around the axis of the cylindrical cartridge, which is formed, for example, by connecting the center of the upper end of the cylindrical structure with the center of the bottom end. In other embodiments, the cylindrical cartridge maintains a fixed position in space, and the cylinder housing rotates around the cylindrical cartridge.

[0098] In certain embodiments, reusable magnets are used to process samples using disposable consumable cylinder cartridges within a sample processing device. The use of reusable magnets reduces waste from each consumable. In certain embodiments, the cylindrical housing is reusable.

[0099] Figure 5 shows a sample preparation system, including a sample preparation cartridge 100 and a cylinder housing 130, according to one embodiment of the present disclosure. In this example, the sample preparation cartridge 100 includes a cylindrical structure 110, a cover 120, a protective cover 140, and a cap 150. The figure also depicts an annular wall 155 of the cylindrical structure and three cavities within the annular wall, forming chambers 160a-160c with three sides open on the annular wall. As seen in the figure, the open sides of each chamber 160a-160c are oriented outward from the cylindrical structure 110. In addition, the open sides of the chambers 160a-160c are enclosed by the cover 120. In Figure 5, the cover 120 is depicted as transparent to help visualize the chambers 160a-160c, although the cover 120 does not need to be transparent. The cover 120 is curved to fit with the outer surface of the annular wall 155, and fluidly seals the open sides of the chambers. The diagram also shows the interconnection 165 between the chambers. As can be seen in the diagram, the interconnection 165 is a channel, which is a recess in the annular wall between the chambers. The diagram also depicts the magnet 170 inside the cylinder housing 130. As can be seen, the magnet 170 is positioned close to the outside of the annular wall 155 of the cylindrical structure 110.

[0100] Figure 6 shows a diagram of the interface boundary between the air phase in the intermediate chamber and the aqueous phase in two chambers adjacent to the intermediate chamber in a cylindrical sample preparation cartridge disclosed herein. Although the figure uses a cylindrical sample preparation cartridge, it should be understood that the depiction also applies to other sample preparation cartridges containing air and aqueous phases, such as a linear sample preparation cartridge; see, for example, Figure 1C. Figure 6 shows a sample preparation cartridge 600 having an air chamber 620 with water-containing chambers 610 and 630 on either side. The framed area includes a first channel 604, the air chamber 620, and part of the second channel 605. During mixing of the PMP and aqueous phase, the first 604 and second 605 channels may be partially filled with the aqueous phase. For example, the lysis buffer present in the water-containing chamber 610 may flow into the first channel 604 during agitation of the sample preparation cartridge. The elution buffer present in the water-containing chamber 630 may flow into the second channel 605 during agitation of the sample preparation cartridge. The presence of air in the intermediate chamber 620 results in the formation of an interfacial boundary at the interface between the aqueous phase and the air phase. The interfacial boundary substantially prevents the aqueous phase from flowing into the air chamber. In some examples, the air chamber includes a reservoir area for accommodating any aqueous phase that may flow into the air chamber, for example, during agitation of the cartridge, and mixing first and second collections of magnetic particles with the sample. A magnet 640 is depicted located outside the sample preparation cartridge. The magnet attracts and holds the magnetic particles and transports them across the liquid-air interface, thereby largely removing the liquid bound to the magnetic particles (e.g., bound to the magnetic particles and / or trapped between the magnetic particles). The magnet then transports the magnetic particles back into the aqueous phase (e.g., elution buffer).

[0101] Automation of the usage method for sample preparation cartridges Certain embodiments also provide a sample preparation cartridge that can be operated using a motor. By automating the motor, the method of using the sample preparation cartridge disclosed herein can be automated. The motor may also be controlled by a computer program, which, when executed by a processor, causes the motor to perform the method of using the cartridge disclosed herein.

[0102] In one particular embodiment, the motor rotates the cylindrical structure in increments of 1.8°.

[0103] In some embodiments, the motor rotates the cylindrical structure back to a predetermined position where, for example, the magnet is positioned close to the dissolution chamber, immiscibility chamber, or elution chamber.

[0104] A motor can be configured to provide only a fraction of a full 360° rotation. For example, a motor can be configured to provide only a rotation of 60° to 120°, preferably 80° to 110°, more preferably 90° to 100°, and most preferably about 90°.

[0105] In some embodiments, a motor can further facilitate the mixing of the contents of the sample preparation cartridge. Such mixing can be carried out by motor-mediated shaking of the sample preparation cartridge. Proper mixing can be provided by controlling the starting position, amplitude, and / or shaking speed. Mixing can reduce sample preparation time and / or improve sample preparation by reducing nonspecific binding and improving homogeneous mixing.

[0106] In certain embodiments, rotating the cylindrical cartridge from a first position to a second position includes rotating the cylindrical cartridge so that the entire span of the dissolution chamber rotates across the magnet. That is, the cylindrical cartridge may be rotated so that the entire lateral span of the dissolution chamber is exposed to the magnet.

[0107] Similarly, in certain embodiments, rotating the cylindrical cartridge from a second position to a third position involves rotating the cylindrical cartridge so that the entire span of the unmixed phase chamber rotates across the magnet. That is, the cylindrical cartridge can be rotated so that the entire lateral span of the unmixed phase chamber is exposed to the magnet.

[0108] The method of the present disclosure may include the additional steps of filling a dissolution chamber with dissolution buffer and paramagnetic particles from a fluid pack contained in a buffer pack, and filling an elution chamber with elution buffer from a fluid pack contained in a buffer pack. In embodiments utilizing a non-air immiscible phase, the step may additionally include filling an immiscible phase chamber with an immiscible phase from a fluid pack contained in a buffer pack.

[0109] In certain embodiments, the fluid is transferred from a fluid pack contained within a buffer pack to a chamber by pressurizing the fluid in the fluid pack and forcing it through channels in the cylindrical structure of the sample preparation cartridge. For example, the fluid may include a dissolution buffer, an immiscible phase, and an elution buffer, some of which contain paramagnetic particles. In some cases, the immiscible phase contains oil.

[0110] When the fluid is transferred from the fluid pack, in certain embodiments, pressure is applied to the fluid in the fluid pack by applying a mechanical force to the cap of a sample preparation cartridge, which has an arm for engaging with the fluid pack. The cap means any convenient mechanical structure having an arm for engaging with the fluid pack. For example, the cap may have a substantially flat base from which the arm protrudes from one side, so that when a force is applied to the flat side of the cap, such force is transmitted along the arm protruding from the base, which then engages with the fluid in the fluid pack, thereby pressurizing the fluid in the fluid pack and forcing the fluid through a channel in a cylindrical structure.

[0111] The method of the present disclosure may include an additional step of transferring the eluted nucleic acid from the elution chamber of the sample preparation cartridge by ejecting the contents of the elution chamber through a drain hole in the chamber. Ejecting the elution chamber can take any convenient form. For example, the sample preparation cartridge may include a plunger assembly, which includes a plunger configured to engage with the elution chamber, which can be automatically triggered to eject the elution chamber when the cylindrical structure is rotated to a specified position.

[0112] In certain embodiments, when the eluted nucleic acid is ejected from the elution chamber, the sample preparation cartridge further comprises a plunger, spring, and trigger working together, and as a result, ejecting the elution chamber involves applying pressure to the trigger to release the tension of the spring, thereby driving the plunger into the elution chamber. In certain embodiments, the cylindrical structure is rotated to a fourth position to allow a mechanical arm to apply pressure to the trigger. In such a case, the trigger may protrude beyond the outer radius of the cylindrical structure. A mechanical arm means any convenient device for use when pressing the trigger. For example, such a mechanical arm may be mounted in a fixed position and arranged to engage with the trigger only when the cylindrical cartridge rotates to a position where the mechanical arm contacts the trigger.

[0113] In a particular embodiment, a sample containing cells is introduced into the lysis buffer by applying pressure to the sample entry component of the sample preparation cartridge, thereby introducing the sample containing cells into the lysis buffer. The sample entry component refers to any convenient structure for surrounding cells such that when force is applied to the structure, pressure is applied to the sample, thereby forcing the sample out of the structure into the lysis chamber of the sample preparation cartridge. [Examples]

[0114] Example 1: Use of air phase for sample preparation Air chambers generally exhibit higher interfacial energy and require much higher pull-through forces (liquid-air penetration forces). This invention provides a solution to the higher overall force requirements for transporting PMP from the aqueous phase to the air phase by adding “helper beads” to assist this boundary transition. These helper beads are typically hydrophilic, larger in size, and / or denser in magnetite, and possess a high magnetic response. Due to their size and larger mass per bead, they can be slightly magnetized in the presence of a permanent magnet. This induced magnetism of the helper beads can assist and accelerate the aggregation of smaller beads in the surrounding solution. While the PMP is functionalized to capture the target analyte, the helper beads do not bind to the target analyte.

[0115] Approximately 800,000 magnetic beads (JSR Scientific MS300) with an average diameter of 2.7 μm were mixed with approximately 16,000 to 32,000 helper beads (Sigma 49664) with an average diameter of 10 μm at a ratio of approximately 1:1 (50% bead mass) per reaction.

[0116] In a separate experiment, approximately 8.2 million magnetic beads (Qiagen MagAttract) with an average diameter of 3.7 μm were mixed with approximately 3,000 to 10,000 helper beads (GE Healthcare) with an average diameter of 100 μm at a ratio of approximately 1:1 (50% bead mass) per reaction.

[0117] Figure 7 shows the results when using air or oil as the immiscible phase. Air transfer required close placement of the outer magnet. Transfer through oil was less sensitive to the magnet placement. When using Qiagen capture beads alone, the maximum separation distance between the magnet and the cartridge, e.g., the cartridge shown in Figure 1D, is 0.5 mm. Beyond this distance, a significant amount of beads are lost at the water-air interface. Since the amount of PMP transferred directly affects the assay sensitivity and the recovered nucleic acid content, an acceptable transfer rate of approximately 90% is expected. A 90% transfer yield is achievable by adding helper beads. Helper beads also allow for magnet placement up to 1.5 mm away from the cartridge. Turbidity of the aqueous phase containing PMP was used as a measure of the PMP transfer percentage. The effect of the immiscible phase (air or oil) on reducing the transfer of the aqueous phase containing PMP was compared using inhibitor carryover.

[0118] Therefore, the above description merely illustrates the principles of this disclosure. Those skilled in the art will understand that various configurations embodying the principles of the present invention and falling within its spirit and scope can be devised, although these are not expressly described or illustrated herein. Furthermore, all examples and conditional statements enumerated herein are intended primarily to assist the reader in understanding the principles of the present invention and the concepts to which the inventors have contributed to the advancement of the art, and should be interpreted not as limitations to such specifically enumerated examples and conditions. In addition, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any elements to be developed that perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments illustrated and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

Claims

1. A method for processing a sample containing or suspected to contain a target analyte, The sample is brought into contact with a first group of magnetic particles and a second group of magnetic particles in the aqueous phase in a first region of the sample processing cartridge, The method includes applying a magnetic force to the magnetic particles to transport the first and second groups of magnetic particles from the aqueous phase in the first region to the air phase in the second region of the cartridge. Here, the first group of magnetic particles is capable of associating with the target analyte, and the second group of magnetic particles is incapable of associating with the target analyte. A method wherein the magnetic particles in the second group are at least twice as large in size as the magnetic particles in the first group.

2. The method according to claim 1, wherein the magnetic particles in the first group have a diameter of 500 nm to 10 μm.

3. The method according to claim 1 or 2, wherein the magnetic particles in the second group have a diameter that is 2 to 20 times the diameter of the magnetic particles in the first group.

4. The method according to any one of claims 1 to 3, further comprising transporting the first and second groups of magnetic particles from the air phase in the second region to the aqueous phase in the third region of the cartridge by applying a magnetic force to the magnetic particles.

5. The method according to claim 4, wherein applying the magnetic force causes the magnetic particles to aggregate in an area in the first region, the area being adjacent to the source of the magnetic force, and transporting the particles to maintain the magnetic force on the aggregated magnetic particles, moving the aggregated magnetic particles to the air phase in the second region of the cartridge, and moving the aggregated magnetic particles to the aqueous phase in the third region of the cartridge.

6. The method according to any one of claims 1 to 5, wherein transporting the first and second groups of magnetic particles includes moving a magnet that generates the magnetic force to different regions of the cartridge.

7. The method according to any one of claims 1 to 5, wherein transporting the first and second groups of magnetic particles includes moving the cartridge or a portion thereof with respect to a magnet that generates the magnetic force.

8. The method according to any one of claims 1 to 5, wherein the cartridge is planar or cylindrical.

9. The method according to claim 8, wherein the cartridge is planar and comprises a first plate positioned spaced apart from a second plate, the first plate and the second plate are held in a stationary position relative to each other, the first plate is a horizontal upper plate and the second plate is a horizontal bottom plate, and the first plate and the second plate are spaced apart from each other by a gap.

10. The method according to claim 8, wherein the cartridge comprises a first plate which is planar and spaced apart from a second plate, the first plate and the second plate which are movable relative to each other such that the plates are held in a configuration which allows them to slide apart, the first plate which is a horizontal upper plate and the second plate which is a horizontal bottom plate, and the first plate and the second plate which are spaced apart from each other by a gap.

11. The method according to any one of claims 8 to 10, wherein the cartridge is planar and does not include a separate chamber, the aqueous phase in the first region is aqueous droplets, the air phase in the second region is air present between the first plate and the second plate, and if present, the aqueous phase in the third region is aqueous droplets.

12. The method according to any one of claims 1 to 9, wherein the first region is a first chamber containing the aqueous phase, and the second region is a second chamber containing the air phase.

13. The method according to claim 12, wherein the first chamber and the second chamber are connected via a first channel, and the pressure difference between the first chamber and the second chamber establishes a liquid-air interface within the first channel.

14. The method according to any one of claims 4 to 9, wherein the third region is a third chamber containing the aqueous phase.

15. The method according to claim 14, wherein the first chamber and the second chamber are connected via a first channel, and the pressure difference between the first chamber and the second chamber establishes a liquid-air interface within the first channel, and the second chamber and the third chamber are connected via a second channel, and the pressure difference between the second chamber and the third chamber establishes an air-liquid interface within the second channel.

16. The method according to claim 1, further comprising transporting the first and second collections of magnetic particles from the air phase in the second region to the aqueous phase in the second cartridge, wherein the transport includes maintaining a magnetic force on the magnetic particles until the second region is connected to the aqueous phase in the second cartridge, and removing the magnetic force so that the magnetic particles are released into the aqueous phase of the second cartridge.

17. The method according to claim 16, wherein the first region and the second region of the cartridge are detachably connected.

18. The method according to claim 16 or 17, wherein the first region is a first chamber in the cartridge, the second region is a transfer plate connected to the aqueous phase in the second cartridge, and the third region is a third chamber in the second cartridge.

19. The method according to any one of claims 1 to 18, wherein the contact includes contacting the sample with a dissolution buffer containing the first and second groups of magnetic particles.

20. The method according to any one of claims 1 to 19, wherein the contact includes placing the sample in the first region, and subsequently introducing the first and second groups of magnetic particles into the first region.

21. The method according to any one of claims 1 to 20, wherein the target analyte comprises cells, viruses, proteins, or nucleic acids.

22. The method according to claim 21, wherein the target analyte includes nucleic acids present in cells or viruses.

23. The method according to any one of claims 1 to 22, wherein the contact results in the destruction of cells or viruses present in the sample, and in each case, the release of nucleic acids present in the cells or viruses.

24. The method according to claim 23, wherein the second group of magnetic particles is incapable of associating with the target analyte, and optionally the target analyte comprises nucleic acids.

25. The method according to any one of claims 12 to 24, wherein the second chamber contains compressed air, and the compressed air is generated by filling the first chamber and the third chamber with an aqueous solution at atmospheric pressure.

26. The method according to any one of claims 12 to 25, wherein the aqueous phase in the third region comprises an elution buffer.

27. The method according to any one of claims 1 to 26, wherein the aqueous phase in the third region contains a washing solution, and the cartridge comprises a fourth region containing air or an immiscible substance and a fifth region containing an elution buffer, wherein optionally the fourth region and the fifth region are chambers.

28. The method according to claim 27, further comprising transporting the first and second groups of magnetic particles from the third region through the fourth region to the fifth region.

29. The method according to any one of claims 1 to 28, wherein the transport includes moving a magnetic field relative to the cartridge while the cartridge remains stationary.

30. The method according to any one of claims 1 to 28, wherein the transport includes moving the cartridge relative to a stationary magnetic field.

31. The method according to any one of claims 1 to 28, wherein the transport includes moving the cartridge and the magnetic field relative to each other.

32. The method according to any one of claims 1 to 31, wherein the contact includes stirring a mixture comprising the sample and the first and second groups of magnetic particles.

33. The method according to claim 32, wherein stirring includes shaking the cartridge.

34. The method according to any one of claims 13 to 33, wherein applying the magnetic force forms aggregates of the first and second groups of magnetic particles, and the aggregates are spatially aligned with the entrance to the first channel.

35. The method according to claim 34, wherein the inlet to the first channel comprises a tapered region that decreases in size from the first chamber to the first channel, facilitating the transport of the aggregate from the first chamber to the second chamber through the first channel.

36. The method according to claim 34 or 35, wherein the aggregate is spatially aligned with the inlet to the second channel.

37. The method according to claim 36, wherein the inlet to the second channel comprises a tapered region that decreases in size from the second chamber to the second channel, facilitating the transport of the aggregate from the second chamber to the third chamber through the second channel.

38. The method according to any one of claims 13 to 37, wherein transporting the first and second groups of magnetic particles from the first chamber to the second chamber of the cartridge by applying a magnetic force to the particles includes arranging a magnet adjacent to the first chamber to cause the formation of an aggregate containing the magnetic particles, the magnet being positioned such that the aggregate is spatially aligned with the inlets to the first and second channels.

39. The method according to any one of claims 1 to 23, wherein the method is semi-automatic.

40. The method according to any one of claims 1 to 23, wherein the step of bringing a sample into contact with a first group of magnetic particles and a second group of magnetic particles in a first area of ​​a sample processing cartridge includes loading the sample into the first chamber of the sample processing cartridge by a user or via a robot, and one or more of the remaining steps are performed automatically by an instrument operably connected to the cartridge.

41. The method according to any one of claims 1 to 40, wherein the ratio of the mass of magnetic particles in the first group to the mass of magnetic particles in the second group is 3:1 to 1:

100.

42. The method according to claim 41, wherein the ratio is 1:1, 2:1, 1:2, 3:1, 1:3, 1:10, 1:30, or 1:100.

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