Patent Publication Number: US-2021178388-A1

Title: Sample preparation system

Description:
RELATED APPLICATION 
     This application claims priority to European Patent Application 19215703.0, filed Dec. 12, 2019, entitled SAMPLE PREPARATION SYSTEM, the disclosure of which is incorporated herein by reference. 
     BACKGROUND 
     In the field of diagnostics there has been a growing need to provide sample preparation devices that can be used in the analysis of a sample from a patient. In particular there has been a growing need for ‘point-of-care’ diagnostic devices that enable a sample to be analysed at the location of a patient to ensure rapid analysis and to improve overall care for the patient. 
     The point-of-care diagnostics market has been growing for several years with the ultimate goal of fulfilling the promise of personalised medicine, and providing the right therapy at the right time for the right patient. Many analytical approaches can be applied to samples, such as molecular diagnostics, chemical analysis, immunoassays and flow cytometry. Whatever the analytical approach, there is a need to be able to supply the sample to the analytical device in a safe and reliable manner. There is furthermore a need to provide a sample preparation cartridge which is small in size and weight, as well as being easy to manufacture and of low cost. 
     One analytical approach which is desired to be implemented in such a point-of-care device is magnetic elution. Magnetic elution is a technique in which a target substance, such as an antibody, is extracted from a sample fluid. 
     Some of the analytical techniques used in such sample preparation systems involve the use of magnetic beads for elution of sample fluids in a sample analysis vessel. Magnetic beads, having the capability to bind to a target substance (e.g. proteins, lipids, DNA, RNA), are introduced into the sample fluid in the vessel. As the vessel is incubated, the desired target substance binds to the magnetic beads. The magnetic beads can then be manipulated using magnets external to the vessel, to separate the target substance from the solvent fluid. Whilst theoretically this approach can allow the target substance to be separated from the solvent, in practice it is difficult to capture all of the magnetic beads in the vessel after elution. Furthermore, sample fluid can become trapped between the magnetic beads leading to low capture efficiency. 
     As a result, current systems have low capture efficiencies and produce low concentrations of the eluent. 
     The present invention seeks to mitigate or address at least some of the above problems. 
     SUMMARY OF THE INVENTION 
     According to an aspect there is provided a sample preparation device comprising a sample preparation vessel and a bead manipulation mechanism for manipulating sample preparation beads in the vessel, the bead manipulation mechanism comprising: one or more magnets arranged to produce a magnetic field for manipulating, in use, one or more beads in the vessel, wherein each magnet is attached to a support frame by a biasing support arranged to support the magnet in contact against an outer surface of the vessel such that the magnet maintains contact with the vessel during relative movement between the magnets and the vessel. 
     By having magnets attached to a support frame by a biasing support, it is possible to provide magnets which can maintain close contact to the vessel even during relative movement of the magnets with respect to the vessel. As a result, the magnets can be moved relative to the vessel whilst ensuring the required proximity to ensure effective engagement of the resulting magnetic field with the magnetic beads in the vessel. Furthermore, by having magnets attached to the support frame the magnets can be moved in relation to the vessel with a single motion of the support frame. The relative motion in this respect may be a motion along the longitudinal axis of the vessel, or it may be a motion along the longitudinal axis of the support frame, or both. The device may be modular and, in particular, the bead manipulation mechanism may itself be a standalone device configured for use with a reaction vessel. 
     Preferably each magnet is provided with its own biasing support, such that each magnet of the one or more magnets is attached to the support frame by a respective biasing support. In examples comprising two or more magnets, each biasing support may be arranged to support the respective magnet in contact against an outer surface of the vessel such that the respective magnet maintains contact with the vessel during relative movement between the respective magnet and the vessel (e.g. as the magnets slide across the surface of the vessel). Thus there may be provided a plurality of magnets attached to the support frame and biased against the vessel by a corresponding plurality of biasing supports. 
     The biasing support or biasing supports may take any form required to provide the necessary resilient force for keeping the magnets in contact with the vessel even when the magnets are moved relative to the vessel. Each biasing support may provide the bias by means of an elastic material, a coil, a spring, or simply by the resilience of a non-elastic material to an equilibrium state. Generally, each biasing support may be laterally flexible. Each biasing support may be biased towards a central axis of the vessel. Preferably, each biasing support may be biased towards a central longitudinal axis of the vessel. Such a support or supports allows the system to have symmetry about the longitudinal axis of the vessel and allows magnets to be positioned about the vessel in a simple and cost-effective manner. 
     Typically, each of the one or more magnets may be attached to a single support frame. The bead manipulation mechanism may further comprise a driving member arranged to cause, in use, movement of the support frame relative to the vessel. When all of the magnets are connected to a single support frame, a driving member can effect movement of all of the magnets in the device by a single movement of the support frame. This reduces the number of moving components required and increases energy efficiency, whilst simplifying the design. The driving member may be arranged to cause movement of any kind to the support frame and magnets. Typically the driving member may be arranged to cause vertical movement along a central longitudinal axis of the vessel. 
     The bead manipulation mechanism may comprise any plurality of magnets positioned at various different orientations with respect to the support frame, and, in use, the vessel. Typically, the bead manipulation mechanism comprises two magnets, positioned in use at laterally opposing sides of the vessel. The lateral direction may be a direction perpendicular to the longitudinal axis of the vessel, or a direction perpendicular to the longitudinal axis of the support frame, or a direction perpendicular to the direction of movement of the bead manipulation mechanism in use. By having magnets arranged to be at laterally opposing sides of the vessel it is possible to provide a symmetrical distribution of magnetic field through the cross section of the vessel. Furthermore it is possible to even out the contact forces of the magnets against the vessel (due to the resilient bias force of the biasing support), as force is applied from opposite sides of the vessel. Symmetry may be maintained using any number of magnets, by configuring the magnets in a particular cross-sectional arrangement. For example, the manipulation mechanism may comprise three magnets arranged as a triangular distribution through the plane of the vessel&#39;s cross section. In another example the manipulation mechanism may comprise four magnets arranged in two opposing pairs around the cross-section of the vessel. Of course, in other examples, the magnets may be configured to be in a non-symmetric arrangement about the cross-section of the vessel. This may be useful for example if a particular directionality is preferred—i.e. if a magnetic bias in one lateral direction is preferred. Alternatively, a preferred magnetic directionality may also be implemented in a symmetric arrangement by using magnets of different strengths. As discussed above, in each of these examples the plurality of magnets (i.e. the two or more magnets) may be maintained in contact with the vessel by a corresponding plurality of biasing supports. Each magnet may be attached to the support from by a respective biasing support, each biasing support being arranged to support the respective magnet in contact against an outer surface of the vessel such that the respective magnet maintains contact with the vessel during relative movement between the respective magnet and the vessel. 
     Whilst the vessel may take any form suitable for containing a sample and magnetic beads, typically the vessel may comprise a top end having an aperture and a bottom end. The bottom end may typically be closed or sealed and impermeable to fluid diffusion. The vessel may be substantially conical, or frustoconical. Typically, such conical or frustoconical vessels may comprise a tapered surface at a bottom end. A tapered bottom end may allow particles and/or fluids of higher density to settle in a concentrated area at the bottom of the vessel. In particular, the tapered bottom end of the vessel may allow magnetic beads and eluent to gather in a concentrated area at the bottom of the vessel. The tapered surface may be straight edge tapers or curved tapers. 
     When the vessel has one or more tapered surfaces, the magnetic beads may be arranged to engage the vessel along the tapered surface(s) of the vessel. In particular, the vessel may have a conical bottom end, with a tapered conical incline leading to a vertex. The beads may engage the vessel along the tapered surface of the vessel so as to ‘slide’ along the surface when the magnets are made to move in relation to the vessel. When the vessel has a conical bottom end, with a tapered incline (or slope) connecting the non-tapered region to a vertex, the magnets may be arranged to move along the inclined surface between the non-tapered region to the vertex, and vice-versa. Movement of the magnets along the tapered surface may cause the lateral separation of the magnets to vary: in particular, in the case of a conical bottom end the magnets may become closer together as they move down the taper (i.e. towards the vertex). 
     The driving member may be operable to drive the support frame to a first configuration in which the magnets are at a bottom end of the vessel, and the driving member may be further operable to drive the support frame to a second configuration in which the magnets are at a top end of the vessel. Alternatively, in the second configuration the magnets may be at a position between the top end and bottom end of the vessel. For example, in the second configuration the magnets may be at an end of a tapered surface of the bottom end, which may not necessarily be the same as the top end of the vessel. 
     The driving member may be further operable to a third configuration wherein the magnets are at a position along the length of the vessel. Alternatively, in the third configuration the magnets may be disengaged from the vessel—i.e. the magnets are not in contact with the vessel. The driving member may be reversibly operable from the first, second or third configuration to any other configuration. 
     In at least one of the driving member configurations, the magnets may be positioned and arranged such that the resulting magnetic field attracts the magnetic beads in the vessel to the bottom of the vessel. Typically, in the first configuration, the magnets may be arranged such that the resulting magnetic field attracts magnetic beads in the vessel to the bottom of the vessel. By having a configuration in which the beads are attracted and gathered at the bottom of the vessel, magnetic beads can be concentrated at the bottom of the vessel to provide a concentrated eluent. In such a case the beads and eluent are gathered at the bottom so as to have a low tide mark, with a low elution volume. Such effects lead to a significantly increased concentration of the eluent particularly when compared to conventional systems. 
     In at least one of the driving member configurations, the magnets may be positioned at a side wall of the vessel. When the magnets are at a side wall, the magnets may be positioned, longitudinally speaking, between a top end and a bottom end of the vessel. Typically, in the second configuration, the magnets may be arranged such that the resulting magnetic field attracts magnetic beads to a side wall of the vessel. The magnets may be arranged so as to attract the beads to opposing sides of the vessel. 
     Of course, the magnets may be arranged to have any of the above mentioned configurations without a driving member, simply by arranging the support frame for example in a suitable position. 
     According to another aspect there is provided a method for manipulating magnetic beads inside a sample preparation vessel, the method comprising the steps of: providing a bead manipulation mechanism comprising one or more magnets arranged to produce a magnetic field for manipulating, in use, one or more beads in the vessel, wherein each magnet is attached to a support frame by a biasing support, the biasing support arranged to bias the magnets toward the vessel; moving the support frame with respect to the vessel so as to cause relative movement of the one or more magnets with respect to the vessel, wherein the support frame is moved with respect to the vessel such that the magnets maintain contact with the vessel due to the bias provided by the biasing support. 
     As it will be appreciated, the method of this further aspect may of course use or implement any combination of features of the first aspect as set out above and apply these features as steps in the method. 
     According to another aspect there is provided a sample preparation device, incorporating any combination of features of the first aspect as set out above, wherein the vessel comprises a fixed section of a pipette component; the device further comprising a moveable pipette arm comprising a pipette tip, the pipette arm being configured, in use, to be moved between a position in which the pipette tip is in sealed engagement with the fixed section of the pipette component and a position in which the pipette tip is positioned away from the fixed section. 
     The device may further comprise one or more additional vessels, wherein at least one additional vessel comprises a fixed section of a pipette component and the pipette arm may be further configured, in use, to be moved between adjacent vessels to engage the pipette tip with the fixed section of pipette component of the vessels. 
     The vessels may be arranged around a central axis of a housing and the moveable pipette arm may be arranged to rotate around the axis and lower towards, or raise from, a desired vessel when it has been rotated to be above the desired vessel in use. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       An example sample preparation device will now be described by way of example with reference to the accompanying drawings, in which: 
         FIG. 1  schematically illustrates an example sample preparation device in one example configuration. 
         FIG. 2  schematically illustrates an example sample preparation device in another example configuration. 
         FIG. 3  schematically illustrates an example device in one example configuration. 
         FIG. 4  schematically illustrates an example device in another example configuration. 
         FIG. 5  schematically illustrates an example driving member of an example sample preparation device in one example configuration. 
     
    
    
     DETAILED DESCRIPTION 
     An example sample preparation device  1  is generally illustrated in an assembled configuration in  FIG. 1 . The example device comprises a sample preparation vessel  10  and a bead manipulation mechanism  20 . 
     The vessel  10  contains a sample fluid  11  to be analysed. The sample fluid  11  generally comprises a target substance in a solvent. In the context of elution techniques and related fields, the target substance and solvent are sometimes referred to as eluate and eluent respectively. The target substance is a substance which is contained or suspended in the solvent and which is desired to be isolated from the solvent. Generally, the target substance is a complex molecule such as a protein or nucleic acid (e.g. DNA, RNA), but can be any substance desired to be extracted or isolated from a solvent. Whilst in this example the fluid  11  is depicted as a liquid, the device and related techniques are equally applicable to gas samples. 
     A plurality of sample preparation beads  12  are contained within the vessel  10 . In the example shown in  FIG. 1  the beads  12  are arranged inside the vessel so as to be submerged in the fluid  11 . The beads  12  are magnetic beads, meaning that they respond when brought into proximity of a magnetic field. In particular, the beads  12  respond to magnetic fields by moving in accordance with the generated magnetic field lines. In this example, the magnetic beads are arranged so as to be attracted by magnets on the bead manipulation mechanism  20  (described in detail below).  FIG. 3  schematically illustrates the vessel  10  in use, containing the sample preparation beads  12  within a sample fluid  11 . The fluid  11  comprises the desired target substance  11  a in a solvent. The vessel  10  is shown in  FIG. 3  in an ‘off’ state, where no magnetic field is applied to the inner volume of the vessel. This can be achieved for example by moving the magnets  22  away from the vessel  10  or by switching off the magnets  22  in the case of electromagnets. 
     The magnetic beads  12  are generally adapted to capture the target substance contained in the fluid  11 . In some examples, the beads  12  are adapted to bind the target substance on their surface. In other examples, the beads  12  are adapted to bind the target substance within their inner volumes, i.e. by providing a porous region or cavity within each bead. In this example, each one of the beads  12  comprises one or more binding receptors disposed on its surface. The binding receptors are arranged to specifically bind to the target substance, for example by having a complementary shape or chemical structure to receive and bind at least a part of the target substance. One or more molecules of the target substance can then be effectively captured on the surface of each magnetic bead  12 . As it will be appreciated, in other examples the magnetic beads  12  can have alternative adaptations to allow them to bind to or capture the target substance. 
     The vessel  10  shown in the example is a container having a bottom end  13 . The bottom end  13  is depicted in  FIG. 1  as being tapered conically from the side surface  14  to a vertex  15 . The taper is a straight edge taper. As well as illustrating the advantage of having such a conically tapered bottom end  13 , the example provides a simple view of how the present device operates. Other shapes for the bottom end  13  are also possible—one notable alternative is a rounded bottom end  13  which are similar to the closed bottom ends of laboratory test tubes. Of course, in other example vessels  10  the bottom end  13  may be tapered differently, or may omit the taper entirely or partially. 
     The bead manipulation mechanism  20  illustrated in  FIG. 1  comprises a plurality of magnets  22  attached to a support rod  21 . Each magnet  22  is attached to the rod  21  by means of a respective flexible biasing support  23 . Thus, as shown in  FIG. 1 , each magnet  22  is attached to the support rod  21  by its own biasing support  23 . 
     Whilst  FIG. 1  illustrates, for simplicity, a cross-sectional view of the mechanism  20  depicting two magnets  22  disposed on laterally opposing sides, it will be appreciated that the mechanism  20  may comprise further magnets outside the plane of the cross-section. For example, the mechanism  20  may comprise a further pair of magnets  22  on laterally opposing sides in the plane perpendicular to the plane of the cross-section (i.e. the plane of  FIG. 1 ). The magnets  22  can be permanent magnets, temporary magnets or electromagnets. The strength of the magnets  22  should be such that the beads  12  can be attracted (or repulsed) to a desired region in the vessel  10  without causing destruction or loss. 
     The support rod is generally rigid and provides structural support for the biasing supports  23  and magnets  22 . The biasing supports  23  in this example are secured to the rod  21  by securing means  25 . In other examples, the biasing supports  23  can be attached to the rod  21  by adhesive or other attachment means. In yet other examples the biasing supports  23  can be integral with the rod  21 —i.e. built as a single continuation of the rod material. 
     The biasing supports  23  are arranged to have a preferential configuration and to have a restoring force which acts to bias the supports  23  in the direction of the preferential configuration. In the example shown, each biasing support  23  has a preferred orientation in which the longitudinal axis of each biasing support  23  is parallel to the axis of the support rod  21 . In other words, the biasing supports  23  are arranged with a restoring action (or force) which biases the supports  23  towards alignment with the support rod  21 . In other examples, the biasing supports  23  are biased with a restoring action toward alignment with an axis (e.g. central longitudinal axis) of the vessel  10 . Whilst the biasing supports  23  can be arranged to prefer any suitable configuration, generally the biasing supports  23  are biased to have a restoring action in the direction of the vessel  10 . The purpose of the biasing supports  23  is to support the magnets against the vessel  10 . That is, the biasing supports  23  are arranged to provide structure so as to provide a biasing (or restoring) action so as to maintain contact with the magnets  22  against the outer surface of the vessel  10 . In other words, each magnet  22  is maintained in contact with the vessel  10  during relative movement between the magnet  22  and the vessel  10  by the corresponding or respective biasing support  23 . 
     The biasing supports  23  support the magnets  22  against the outer surface of the vessel  10  such that, when the mechanism is moved in relation to the vessel  10 , the magnets  22  maintain contact against the vessel  10  surface.  FIG. 2  shows the mechanism  20  in a configuration where the support rod  21  has been moved to a position in which the magnets  22  are at a vertex  15  of the tapered bottom end  13  of the vessel  10 . It can be seen that, due to the resilience of the biasing supports  23 , the magnets  22  are in contact with the surface of the vessel  10 . The mechanism  20  can be moved from the configuration of  FIG. 2  to the configuration of  FIG. 1  by advancing the support rod  21  toward the vessel. The axis of advancement is aligned with the central axis of the vessel  10 . It will be appreciated that, due to the resilient biasing action of the biasing supports  23  the magnets  22  slide up the outer surface of the vessel, and maintain contact with the vessel  10  throughout the sliding motion. 
     In use, the vessel  10  is loaded with a sample to be analysed, along with magnetic beads  12 . Normally, the sample is a fluid  11  containing a target substance to be isolated from the rest of the fluid  11 . The beads  12  may be loaded into the vessel  10  first and then mixed with the sample. Alternatively the sample may first be loaded into the vessel and then mixed with the beads  12 . At this stage, the vessel  10  generally resembles the example illustrated at  FIG. 3 , where the sample in vessel  10  comprises the beads  12  and target substance distributed within the fluid  11 . 
     The beads  12  are then to be mixed around the sample so that the target substance can bind to the beads  12 . This can be achieved by an external mixing method (i.e. by stirring, rotating, sliding, tilting or shaking the vessel  10 ). Alternatively, the beads  12  can be mixed around the sample by using the magnets  22  of the bead manipulation mechanism  20 . 
     The bead manipulation mechanism  20  is employed by bringing the magnets  22  in proximity to the vessel  10 . In one example, the mechanism  20  engages the vessel  10  by advancing the magnets  22  to a bottom end  13  of the vessel  10  (as shown in  FIG. 2 ). In this position, the biasing supports  23  are substantially straight (i.e. not in flexion, and substantially parallel to the support rod  21 ) and the magnets  22  can attract the beads  12  to the bottom of the vessel  10 . In alternative examples, the magnets  22  may not be strong enough to attract all of the magnets present in the vessel  10  to the bottom of the vessel in which case the magnets  22  (and the resulting magnetic field) can be moved around the vessel  10  to bring all the magnetic beads  12  into proximity. 
     As the bead manipulation mechanism  20  is advanced upwards, the magnets  22  are slid up the surface of the vessel  10  and, due to the tapered bottom end of the vessel  10 , the magnets are laterally displaced. Because of this lateral displacement, the biasing supports  23  become laterally displaced at one end causing flexion. The flexion of the biasing supports  23  is counteracted by the resilient restoring force in the supports  23  to maintain the magnets  22  in contact with the vessel  10  surface, whilst allowing the magnets  22  to follow the outer contour of the vessel  10 . 
     As the support rod  21  advances towards the vessel  10 , the magnets  22  part from each other to match the outer surface of the vessel  10 . This stage is illustrated in  FIG. 1 . In this state, the magnets  22  are no longer at the bottom vertex  15  of the vessel and are now on the side walls of the vessel  10 . The magnets  22  attract the magnetic beads  12  within the vessel to the interior side wall(s) of the vessel  10 . 
     It can thus be seen that by a simple movement of the support rod  21  ‘up’ and ‘down’ with respect to the vessel  10 , the mechanism  20  is able to control the position of magnetic beads  12  within the vessel. In particular, the beads  12  can be easily and precisely manipulated from various positions on the side walls of the vessel  10 , to the bottom vertex of the vessel  10 . One particular advantage of the system described herein is that the beads can be easily and efficiently manipulated, by moving the mechanism  20  to the configuration shown in  FIG. 2  for example, to the bottom of the vessel, which results in a low tide mark and a highly concentrated eluent at the bottom of the vessel  10 . 
     By moving the mechanism  20  into a configuration in which the magnets  22  are at the bottom of the vessel, the magnets beads  12  in the vessel can be gathered into a ‘clump’ at the bottom inner surface of the vessel  10 . This configuration can be seen in  FIG. 4 . With most or all of the magnets gathered in one location, the sample fluid (solvent) can be aspirated out of the vessel  10  so as to leave the magnetic beads  12  which are left in place at the bottom surface of the vessel  10 . Aspiration can be done by suitable means, for example by a pipette or pipette system provided with the device. The beads  12  may remain at the bottom simply by gravity or surface tension, or the beads  12  may be held in place at the bottom by means of the magnets  22  on the manipulation mechanism  20 . As it can be seen from  FIG. 4 , the present invention allows the beads  12  to be collected in a highly concentrated region with a low tide mark so as to leave a highly concentrated eluate. A further advantage is that, due to the low tide mark, only a relatively small volume of fluid is needed for the elution step, which further increases the concentration of the eluent. 
     Another useful function of the device is that the bead manipulation mechanism  20  can be moved to move the magnets  22  up the side of the vessel  10  so as to capture the beads  12  and move the clumped magnetic beads  12  up and against a side wall of the vessel  10 . By moving the magnetic beads  12  up at a side wall, residue trapped between or within the beads can be drained to the bottom of the chamber where it can be easily and effectively removed (for example by aspiration by pipette). 
     Relative movement of the manipulation mechanism  20  with respect to the vessel  10  can be effected manually or automatically. It will be appreciated that, in order to provide relative movement, the vessel  10  can be moved in relation to the mechanism  20  or the mechanism  20  can be moved in relation to the vessel  10 , or both. The system can be configured with a driving member  30 , as shown in  FIG. 5 , to effect the relative movement between the vessel  10  and the bead manipulation mechanism  20 . In the example shown the driving member  30  comprises a vessel holder for holding a vessel  10 . The driving member  30  also comprises a housing  32  which mounts the bead manipulation mechanism  20 . The vessel holder  31  is mounted to the driving member housing  32  by a moveable connection, such that holder  31  can move in relation to the housing  32 . This can be achieved for example by a piston mechanism connected to a motor. 
     In use, a vessel  10  is located in the holder  31 . The vessel  10  may be integral with the holder  31  or alternatively the vessel  10  may be built as a standalone component and placed (and secured) into the holder  31 . A bead manipulation mechanism  20  as described above is provided in or on the housing  32 . The driving member  30  operates by moving the holder  31  in relation to the housing  32  so as to cause relative movement between the vessel  10  and the mechanism  20 . The driving member  30  can be programmed to work synchronously with other functions of the sample preparation device. For example, the driving member  30  may co-operate with a pipetting or aspirating device to allow automated or programmed aspiration of the sample fluid. 
     The bead manipulation mechanism can be utilised in a device comprising a plurality of vessels. In such a case, each vessel may be fitted with a bead manipulation mechanism at the vessel, or alternatively one more or manipulation mechanisms can be configurable between the vessels to provide functionality to more than one vessel in the device. Where the device comprises a pipette tip, the pipette tip may be moveable between the pluralities of vessels. The pipette can be formed of a tip extending from a central axis, around which the vessels are distributed, and a fixed portion positioned at the vessel. The pipette tip may be rotatable about the central axis to engage the fixed potion of each vessel. 
     As will be appreciated from the above, the present invention, by providing an innovative bead manipulation mechanism which can simply and effectively manipulate beads inside a sample analysis vessel without complex movement, enables the provision of a sample analysis device which is compact, inexpensive and produces a significantly improved ability to provide purified and concentrated target substances without risk of inhibition. Furthermore, due to the efficient and effective manipulation of beads inside the sample the present invention significantly increases the magnetic bead capture rate which speeds up any analysis and testing carried out with the device.