WO2004011146A1 - Dispositif et procede de manipulation de particules magnetiques - Google Patents

Dispositif et procede de manipulation de particules magnetiques Download PDF

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Publication number
WO2004011146A1
WO2004011146A1 PCT/NO2003/000261 NO0300261W WO2004011146A1 WO 2004011146 A1 WO2004011146 A1 WO 2004011146A1 NO 0300261 W NO0300261 W NO 0300261W WO 2004011146 A1 WO2004011146 A1 WO 2004011146A1
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WO
WIPO (PCT)
Prior art keywords
pipette
fluid
magnetic particles
hgms
matrix
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Application number
PCT/NO2003/000261
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English (en)
Inventor
Andrew T. Campbell
Original Assignee
Campbell Andrew T
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Campbell Andrew T filed Critical Campbell Andrew T
Priority to AU2003248518A priority Critical patent/AU2003248518A1/en
Publication of WO2004011146A1 publication Critical patent/WO2004011146A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
    • B01F31/65—Mixers with shaking, oscillating, or vibrating mechanisms the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
    • B01F31/65—Mixers with shaking, oscillating, or vibrating mechanisms the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
    • B01F31/651—Mixing by successively aspirating a part of the mixture in a conduit, e.g. a piston, and reinjecting it through the same conduit into the receptacle
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/025—High gradient magnetic separators
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • a method and device is provided for the efficient mixing, separation, collection and washing of magnetic particles from fluids .
  • IMS immuno-magnetic separation
  • IMS includes a reaction phase to promote complex formation between the magnetic particles and the target entity, generally facilitated by mixing the magnetic particles in a tube or vessel containing the target entity within a test medium.
  • the magnetic particles are subsequently isolated using a magnetic field gradient, for example by the application of a magnet to the side of the tube or vessel to effect localized concentration, magnetizing a magnetizable matrix within the tube or vessel to immobilize the magnetic particles on the magnetizable matrix or move the magnetic particles to an area distinct from the test medium, thereby effecting isolation.
  • the magnets used include both permanent and electromagnets. During this magnetic isolation step, unwanted material is substantially removed from the target entities, with almost complete removal being effected using sequential wash steps with an aqueous buffer of defined composition.
  • the isolated and purified target entities can then be readily processed further, depending on the application.
  • This has included the subsequent labelling, wash and detection steps described for a range of immunoassays (including enzyme linked, radioactive, fluorescent or luminescent) , nucleic acid detection (e.g. PCR) , as well as the isolation of cells, viruses, proteins, prions or other targets for further use, manipulation or detection.
  • immunoassays including enzyme linked, radioactive, fluorescent or luminescent
  • nucleic acid detection e.g. PCR
  • IMS using negative selection has also been described. In this procedure, the unwanted material is bound to the magnetic particles and removed from the desired target entities when magnetic separation is effected.
  • the magnetic particles that have been described for such applications include mono-, and poly-disperse or cluster- type particles or aggregates, with both monomodal and polymodal size distribution employing spheres, flakes or other solid phases as the base matrix.
  • the sizes of the magnetic particles have been described from 10 nm to 10 mm, but are more commonly 50 nm to 5 ⁇ m.
  • IMS has many advantages over other separation or isolation procedures, including speed of reaction, efficiency, retention of viability or structure of the target and the theoretical application to automation.
  • the speed of reaction and efficiency of the system is generally considered to be a function of the surface area available for interaction with the target and the advantageous mixing kinetics of the magnetic particles. For this reason, theoretically, smaller (increased surface area available for interaction) monodisperse, non-aggregated particles with a density approaching unity are to be preferred, although, in practice, this has been demonstrated not to be case.
  • the improved viability or structural retention of the target is considered to be a consequence of the gentler magnet based isolation over other techniques like centrifugation or column based procedures.
  • the automated IMS systems previously described often require the accurate placing of the magnet. The closer the magnet is to the sample, the faster the quantitative isolation of the magnetic particles. This can be technically complex. It is an attractive feature to continually move or agitate the magnetic particles throughout the procedure using agitators, rods, alternating electromagnets or moving magnets that often require highly specialised equipment.
  • the IMS automates described to date are also generally designed to process a fixed, or relatively narrow range of volumes. This is especially the case when rods are used for mixing or the separation is conducted within a pipette tip. These systems have been, in general, designed with a single application in mind, e.g. to retrofit existing high-throughput robotic workstations or for processing a single, fixed volume.
  • IMS automates can be readily designed to prevent contamination of the target entity with other samples being co-processed, or from extraneous material and will be safer for the operators when processing pathogenic or infections material (e.g. specific microorganisms).
  • pathogenic or infections material e.g. specific microorganisms
  • the device comprises a pipette with a larger diameter flexible or movable walled "bulb" area, that preferably contains a high gradient magnetic separation (HGMS) matrix which can be compressed, pushed or squeezed to effect fluid movement within the system that is connected or integral to said pipette, a magnet, a device for compressing, pushing or squeezing the larger diameter flexible or movable walled bulb area, and a device for moving the magnet toward the pipette and away from it.
  • HGMS high gradient magnetic separation
  • FIG. la & lb Device for mixing, separating and washing magnetic particles.
  • FIG. 2a, 2b & 2c HGMS device for mixing, separating and washing magnetic particles.
  • FIG. 3 Device for concentrating and collecting magnetic particles .
  • FIG. 4 Method for labelling and detecting specific entities using the device.
  • FIG. 5 A description of an automated device of the invention.
  • An objective of the invention is to provide a device and a method for mixing, separating and washing magnetic particles which does not require pumps, valves, mixing rods or separate suction or pressure facilities to manipulate the fluid containing the suspension of magnetic particles.
  • the device comprises a pipette with a larger diameter flexible-walled bulb area which can be compressed, pushed or squeezed, connected or integral to said pipette, a magnet, and a device for moving the pipette toward and away from said magnet, or a device for moving the magnet toward and away from said pipette to enable the magnetic particles to be deposited or immobilized within the larger diameter flexible-walled bulb area of said pipette.
  • a preferred embodiment is an apparatus in accordance with the invention comprising a pipette with the larger diameter flexible-walled bulb connected to said pipette as an integral part of the pipette, however pipette/bulb arrangements that are manufactured as separate units, with assembly post-manufacture, are also envisaged in the present invention.
  • Pipettes with a cylindrical cross- section are preferred, with a single opening at the end of the pipette longitudinal extension tip. It is also possible to use pipettes that have areas with rectangular cross- sections. It is essential to the invention that at least one retaining wall of the larger diameter bulb area of the pipette is flexible or movable and the interior of the pipette can be penetrated by a magnetic field, with plastic usually being employed as the material.
  • the fluid held in the pipette is substantially contained within the larger volume flexible- walled bulb area where separation of the magnetic particles is achieved.
  • the interior of the pipette/bulb arrangement should not have any recesses or edges as this may interfere with the quantitative release of magnetic particles separated in the pipette.
  • Pipettes that have a uniform inner diameter over their longitudinal extension are suitable; it can however be advantageous to have pipettes with tapered ends.
  • the inner wall of the pipette/bulb arrangement should be smooth. It is also possible to use pipettes whose inner walls are provided with a non-adherent coating, for example, a silane coating.
  • the pipette/bulb arrangement is produced by standard blow moulding techniques, the whole unit being produced in plastic that can be discarded after use .
  • Magnetic particles are understood to be particles that are attracted by means of a magnetic field. Hence, the magnetic particles can themselves be magnetized.
  • Preferred materials are paramagnetic materials, i.e., those that exhibit only a minor remanence (i.e., magnetism remaining after the magnetic field has been removed) .
  • the material of the particles can be a compound material, e.g. a matrix that contains magnetically attractable particles. Magnetically attractable materials are, for example, iron, iron oxide, nickel, cobalt, or chromium oxide.
  • One or several particles of this material can be embedded in a matrix.
  • the matrix can consist of a multitude of materials, e.g. organic or inorganic polymers.
  • the larger diameter bulb is first compressed then inserted into the solution containing the magnetic particles.
  • the compression is then controllably released to draw the fluid into the body of the pipette.
  • the magnet is located at an external wall of the larger diameter bulb area of the pipette. This will allow collection of the magnetic particles at the inner wall of the larger diameter bulb area of the pipette.
  • An advantage of this invention is that the magnet will automatically come into direct contact with the pipette bulb when the pipette bulb is compressed as the magnet operates as at least one containment wall of the device that retains the pipette bulb. Therefore in this invention, it is not necessary to have a fine adjustment of the distance between magnet and pipette bulb.
  • the surface of the magnet located in the vicinity of the pipette bulb should have a cross section that is essentially not smaller than the diameter of the pipette bulb.
  • the magnet surface should be planar for the pipette bulb, however magnet geometry' s that match the profile of the pipette bulb can be employed and more than one magnet can be employed.
  • Magnets for separating the magnetic particles can be electrical magnets as well as permanent magnets. Permanent magnets are preferred as the alloys commonly available today exhibit a higher magnetic force per magnet area and do not generate heat during operation, which could interfere with the system. Strong permanent magnets exhibit a magnetic flow that is sufficient to readily separate most magnetic particles from a suspension with a thickness of up to a few centimetres.
  • the magnets used preferably have the form of a block magnet. It is, however, also possible to use magnets with various other shapes or geometry' s .
  • the magnet can be fixed in its position inside the apparatus or attached to a device for moving it spatially.
  • a device in accordance with the invention comprises a device for moving the magnet toward the pipette.
  • the invention is also intended to encompass an embodiment where the pipette is moved towards the magnet.
  • a preferred embodiment of the invention uses a fluid permeable magnetizable high gradient magnetic separation (HGMS) matrix located within the larger diameter flexible- walled bulb area of the pipette. This embodiment is particularly suitable for application to the smaller magnetic particles for example, ferrofluids .
  • the magnetizable HGMS matrix can be secured in position within the pipette bulb area or can be discharged within pipette bulb area. The orientation of the HGMS matrix should permit the compression of the pipette bulb.
  • the magnetizable HGMS matrix can be made of materials such as steel wool, metal-coated fibres, and metal spheres, plates, bars, filings or wires.
  • the magnetizable HGMS matrix is constructed in such a fashion which creates substantially homogeneous fluid flow during the procedure and can either be a rigid production or malleable in organization.
  • the construction of said magnetizable HGMS matrices are well known to one skilled in the art and will not be covered in more detail herein.
  • the HGMS matrix is located within the base of the larger diameter flexible-walled bulb area of the pipette. This configuration will ensure that it is imperative that the fluid containing the magnetic particles passes through the magnetizable HGMS matrix.
  • a known disadvantage of column based HGMS systems can occur when processing test media that contain a high level of particulate material, for example, water sample concentrates, food or feed washings or other environmental material and when manipulating matrices containing aggregates, large cell types or viscous polymers.
  • the magnetizable HGMS matrix has a propensity to become blocked thus obstructing fluid flow through the HGMS matrix or it can be difficult to recover the magnetic particles from the HGMS matrix.
  • a solution to these disadvantages is an advantage conferred by the present invention.
  • the fluid flow is readily and controllably repeatedly reversed, i.e. back-flushed, during the operation, thus minimizing these negative effects experienced for the column based HGMS matrix systems.
  • Another preferred embodiment of this invention locates the magnetizable HGMS matrix in the middle of the larger diameter flexible-walled bulb area of the pipette.
  • a significant advantage of this embodiment is that the fluid suspension containing the magnetic particles is not forced to pass through the HGMS matrix as occurs for column separation systems. This minimizes non-specific entrapment of extraneous material, retains flow, reduces shear forces and facilitates the collection of the magnetic particles at the end of the procedure.
  • To magnetize the HGMS matrix the magnet is introduced at the outer wall of the pipette bulb in such a fashion that the magnet induces a magnetic flux within the HGMS matrix, the magnetic flux thereby magnetizing the HGMS matrix, to which the magnetic particles will be attracted and immobilized.
  • the magnet is attached at a place adjacent to the larger diameter flexible-walled bulb area pipette wall in a configuration that will induce maximum magnetic flux within the HGMS matrix contained within the pipette bulb.
  • This method will effect the efficient mixing of magnetic particles in a fluid.
  • This method will effect the efficient mixing of target entities or labelling reagents in a fluid in the presence of, or through an HGMS matrix containing immobilized magnetic particles.
  • volume contained within the pipette body does not solely govern the volume that can be mixed.
  • the volume that can be effectively contained in a vessel will be the maximum volume capacity of the system.
  • the volume drawn into the pipette bulb would be a minimum of 5% to 10% of the total volume of the system, preferably at least 25% to effect rapid and efficient mixing.
  • the pipette should not draw air during the cycle. Therefore, the diameter and length of the longitudinal extension of the pipette will govern the minimum volume of the system.
  • the pipette bulb compression can be readily and accurately controlled for the appropriate volumes (i.e. little compression for small volumes, maximum for large) . Devices that can process ⁇ 100 ⁇ l to >1000 ml can therefore be readily designed using this invention. Diluting the sample in an appropriate buffer can process smaller volumes. Larger volumes can be processed with increasing time and by employing larger volume containment vessels and pipettes with bulbs that retain a larger volume of fluid.
  • the invention further addresses a method for washing magnetic particles comprising the following steps:
  • the magnet is attached at a place adjacent to at least one wall of the larger volume flexible-walled bulb area of the pipette.
  • the pipetting procedures are carried out such that the total volume of fluid can be ejected from the pipette, if required. This is done by only using maximum compression on the pipette bulb during the eject cycle.
  • the fluid from which the particles have been removed is ejected from the pipette.
  • a second fluid is drawn into the pipette to wash the magnetic particles. This can be done while the magnet is still at the wall of the pipette bulb or, if this is not desired, the magnet can be moved away from the pipette bulb prior thereto.
  • the separated magnetic particles are washed in the flow entering the pipette bulb; in the second case the particles are at least partly mixed in the flow entering the pipette bulb.
  • the fluid in which the magnetic particles are suspended, and the washing fluid may be water or an inert fluid.
  • the fluids contain surface-active agents and/or blocking agents as this increases the washing effect.
  • the particles can be ejected directly with the fluid. If more extensive washing procedures are required, the fluid can be ejected from the body of the pipette while the magnet is still at the wall of the pipette bulb, and further washing fluids introduced according to the already described procedure. Alternatively, the particles can be suspended in the washing fluid according to the already described procedure and be again deposited at the wall of, or immobilized on the magnetizable HGMS matrix contained within the pipette bulb.
  • the efficiency of a washing procedure depends on how homogeneous the magnetic particles are suspended in the washing fluid. Hence it is preferred, particularly when not using an HGMS matrix, to resuspend the magnetic particles in the washing fluid.
  • the described methods for resuspension can also be applied to eject the particles together with the fluid from the pipette .
  • the invention also encompasses a method to concentrate the magnetic particles into a small volume following capture of the magnetic particles in the larger diameter flexible- walled bulb area of the pipette.
  • This comprises a magnet placed at the wall of the pipette towards the tip of the longitudinal end of the pipette, near the opening that the suspension is introduced into the pipette.
  • the fluid from which the particles have been removed is ejected from the pipette.
  • a final system or storage fluid is drawn into the pipette with the magnet moved away from the pipette prior thereto, to resuspend the particles, with fluid oscillation to effect complete resuspension of the particles as required.
  • a magnet is placed at the wall of the pipette towards the tip of the longitudinal end of the pipette before controllably ejecting the fluid from the pipette.
  • the magnetic particles will be concentrated at the inner wall or on a suitable magnetizable HGMS matrix, as described above, at the tip of the longitudinal extension of the pipette.
  • the fluid can be repeatedly, controllably drawn into and expelled from the pipette to effect complete concentration of the magnetic particles.
  • These concentrated particles can then be collected in a suitable system or storage fluid at the required volume using the procedures described above.
  • the invention also addresses a method for carrying out an analysis comprising the steps: Providing a suspension of magnetic particles in a first fluid and either incubating the suspension with an analyte prior to transferring the reaction mixture to a pipette/bulb assembly and moving a magnet to the pipette bulb, or first immobilizing the magnetic particles onto a magnetized HGMS matrix prior to the introduction of the analyte into the pipette/bulb assembly.
  • the fluid phase is ejected from the body of the pipette and a system fluid transferred into the pipette/bulb assembly, then moving the pipette away from the magnet, ejecting the system fluid together with the particles in a receiving container, and determining the analyte concentration based on the specific properties of the system fluid or the particles.
  • Detection methods can include the use of fluorescent, luminescent, enzyme, radio, phosphorescent, spin, dye, nucleic acid, nucleic acid analogue, chelating or heavy metal labels. These assays are known from prior art and their detailed description is therefore omitted here.
  • a system fluid instead of a washing fluid is drawn into the pipette.
  • Said system fluid can either be water or an inert fluid containing detergents, reagents, or auxiliary substances.
  • an active reagent e.g. an antibody carrying a label (e.g. an enzyme, ruthenium label) .
  • the bound analyte is detected after it has been washed again with conventional washing solutions.
  • the analyte can be detected via a colour reaction, for example.
  • the particles are separated in a measurement cell where a measuring signal (e.g. colouration, fluorescence) is generated.
  • a measuring signal e.g. colouration, fluorescence
  • Quantification of the label can also be conducted within the body of the pipette by incorporating suitable optics or other detection devices in the device.
  • FIG. la shows a device of the invention for mixing magnetic particles in a fluid.
  • the top part (1) of the pipette is a cylindrical flexible-walled body with which the longitudinal extension part (2) of the pipette is an integral part.
  • the complete unit is a disposable plastic Pasteur pipette supplied by Labtech International, UK.
  • a compressing device with piston (3) and non-magnetic plate (4) is directly in contact with the top part (1) of the pipette.
  • a fluid (5) containing magnetic particles can be expelled from the pipette through the longitudinal extension (2) into a sample container (6) .
  • FIG. lb further shows a device of the invention for separating and washing the magnetic particles. It is the same Pasteur pipette used described in figure la with a top part (1) and an integral longitudinal extension part (2) .
  • the compressing device with piston (3) and magnet (4) is directly in contact with the top part (1) of the pipette.
  • the fluid (5) can be expelled from the pipette by moving piston (3) towards magnet (4) .
  • the sample container (6) containing the fluid (5) is removed from the device and replaced with a wash container (8) containing a wash solution (9) .
  • the magnet (4) is removed and the compression on the top part (1) of the pipette is released to draw the wash solution into the pipette and resuspend the magnetic particles (7) in the wash solution (9) .
  • the magnet (4) can be immediately replaced and the magnetic particles (7) immobilized to the inner wall of the top part (1) adjacent to the magnet (4) according to the procedure described above.
  • the procedure for mixing described in figure la should be followed, prior to magnetic capture as described above.
  • the magnetic particles (7) are immobilized at the inner wall of the pipette adjacent to the magnet (4) as previously described.
  • the wash solution (9) is expelled from the pipette by moving piston (3) towards magnet (4) .
  • the wash container (8) containing the wash solution (9) is removed from the device and replaced with a wash container (8) containing fresh wash solution (9).
  • the magnet (4) is removed and a non-magnetic plate inserted (not shown) and the compression on the top part (1) of the pipette is released to draw the wash solution (9) into the pipette and resuspend the magnetic particles (7) in the fresh wash solution (9).
  • the piston (3) is moved towards the non-magnetic plate, and the wash solution (9) containing magnetic particles can be expelled from the pipette through the longitudinal extension (2) into a suitable collection container (not shown) .
  • FIGS. 2a & 2b show devices of the invention for separating and washing the magnetic particles using a magnetizable HGMS matrix.
  • the HGMS matrix in figure 2b is malleable in organization.
  • Both figures utilize a similar Pasteur pipette used described in figure la with a top part (1) and an integral longitudinal extension part (2), but includes a magnetizable HGMS matrix (7) within the top part (1) of the pipette.
  • the compressing device with piston (3) and magnet (4) is directly in contact with the top part (1) of the pipette.
  • the magnet is placed such that it will magnetize the magnetizable HGMS matrix (7) .
  • the fluid (5) is expelled from the pipette by moving piston (3) towards magnet (4).
  • the sample container (6) containing the fluid (5) is removed from the device and replaced with a wash container (8) containing a wash solution (9) .
  • the wash solution (9) can be repeatedly passed through the magnetized HGMS matrix (7) onto which the magnetic particles are immobilized by controllably moving the piston (3) towards and away from the magnet (4) to effect the sequential compression on the top part (1) of the pipette, thus inducing the wash solution to flow through the magnetized HGMS matrix (7) .
  • the wash solution (9) is expelled from the pipette by moving piston (3) towards magnet (4) and the wash container (8) containing the wash solution (9) is removed from the device and replaced with a wash container (8) containing fresh wash solution (9).
  • the magnetic particles immobilized on the magnetized HGMS matrix (7) are collected from the HGMS matrix (7) by releasing the compression on the top part (1) of the pipette to draw the fresh wash solution (9) into the top part (1) of the pipette.
  • the magnet (4) is removed and replaced with a non-magnetic plate (not shown) before moving the piston (3) towards the non-magnetic plate (not shown) .
  • the wash solution (9) entering the magnetizable HGMS matrix (7) from the top part (1) of the pipette resuspends the magnetic particles that were immobilized on the magnetizable HGMS matrix (7).
  • the wash solution (9) containing magnetic particles can be expelled from the pipette through the longitudinal extension (2) into a suitable collection container (not shown) . If required, partial piston compression and release (flow agitation) can be effected to facilitate the complete resuspension of the magnetic particles from the magnetizable HGMS matrix (7) before expelling the wash solution (9) from the pipette.
  • FIG. 2c shows a device of the invention for separating and washing the magnetic particles from fluids that have a high particulate content using a magnetizable high gradient magnetic separator. It is a similar pasteur pipette used described in figure 2a with a top part (1) and an integral longitudinal extension part (2) and a magnetizable HGMS matrix (7) contained within the top part (1) of the pipette.
  • the compressing device with piston (3) and a nonmagnetic plate (4) is directly in contact with the top part (1) of the pipette.
  • the magnet (8) is placed such that it will magnetize the magnetizable HGMS matrix (7) .
  • the piston (3) is mechanically connected to a motor, therefore can be controllably oscillated to effect fluid movement between the top part (1) of the pipette and the sample container (6), thereby repeatedly passage the fluid (5) containing magnetic particles through the magnetized HGMS matrix (7) and effect the efficient collection of the magnetic particles onto the magnetized HGMS matrix (7) from the fluid (5) .
  • Larger particulate material will not enter the magnetizable HGMS matrix (7) and therefore does not have the potential to block the magnetized HGMS matrix (7) .
  • the fluid (5) is expelled from the pipette by moving piston (3) towards the non-magnetic plate (4) .
  • the sample container (6) containing the fluid (5) is removed from the device and replaced with a wash container (9) containing a wash solution (10) .
  • the magnet (8) is retained in position and the compression on the top part (1) of the pipette is released to draw the wash solution (10) into the pipette and into the magnetized HGMS matrix (7) on which the magnetic particles are immobilized.
  • the wash solution (10) can be repeatedly passed through the magnetized HGMS matrix (7) onto which the magnetic particles are immobilized by controllably moving the piston (3) towards and away from the non-magnetic plate (4) to effect the sequential compression on the top part (1) of the pipette, thus inducing the wash solution (10) to flow through the magnetized HGMS matrix (7) .
  • the wash solution (10) is expelled from the pipette by moving piston (3) towards the non-magnetic plate (4) and the wash container (9) containing the wash solution (10) is removed from the device and replaced with a wash container (9) containing fresh wash solution (10).
  • the magnetic particles immobilized on the magnetized HGMS matrix (7) are collected from the HGMS matrix (7) by releasing the compression on the top part (1) of the pipette to draw the fresh wash solution (10) into the top part (1) of the pipette.
  • the magnet (8) is removed from the device before moving the piston (3) towards the non-magnetic plate (4) .
  • the wash solution (10) entering the magnetizable HGMS matrix (7) from the top part (1) of the pipette resuspends the magnetic particles that were immobilized on the magnetizable HGMS matrix (7) .
  • the wash solution (10) containing magnetic particles can be expelled from the pipette through the longitudinal extension (2) into a suitable collection container (not shown) .
  • partial piston compression and release flow agitation
  • a restricted volume of fluid can be processed by not completely releasing the compression on the top part (1) of the pipette.
  • FIG. 3 shows a device of the invention for collecting and concentrating magnetic particles in a fluid after processing and separating magnetic particles with one of the devices described in figures 1 & 2.
  • a compressing device with piston (3) and non-magnetic plate (4) is directly in contact with the top part (1) of the pipette.
  • a fluid (5) containing magnetic particles can be expelled from the top part (1) of the pipette through the longitudinal extension (2) into a fluid container (6) containing the residual fluid (7).
  • magnetic particles (8) that were suspended in the fluid (5) contained within the top part (1) of the pipette have been deposited at the inner wall of, or on a HGMS matrix within the longitudinal extension (2) of the pipette adjacent to the magnet (9) .
  • the tip at the end of the longitudinal extension (2) of the pipette can be inserted into the residual fluid (7) contained within the fluid container ( ⁇ ) and partial piston compression and release can be effected to repeatedly pass the fluid (5) containing the magnetic particles by the magnet (9) to facilitate the complete collection of the magnetic particles.
  • the fluid is expelled from the pipette by moving piston (3) towards the non-magnetic plate (4) and the fluid container (6) containing the residual fluid (7) is removed from the device and a collection or reaction container (not shown) containing a system or storage fluid is inserted into the device.
  • the magnet (9) is removed from the wall of the longitudinal extension (2) of the pipette, before partially releasing the piston (3) compression on the top part (1) of the pipette to draw the system or storage fluid (7) into the longitudinal extension (2) of the pipette sufficient to resuspend the magnetic particles from the wall or HGMS matrix of the longitudinal extension (2) of the pipette.
  • the storage or system fluid can then be expelled from the pipette into the collection container by moving piston (3) towards the non-magnetic plate (4).
  • FIG. 4 shows a possible method of processing the magnetic particles for the detection of a specific target entity bound to the magnetic particles using the device of the invention described in figure 3.
  • FIG 4a fluid is expelled from the pipette by moving piston (3) towards the non-magnetic plate (4) and the fluid container (6) containing the residual fluid (7) is removed from the device and a fluid container (10) containing a system fluid (11), is inserted into the device.
  • FIG 4b & 4c magnet (9) is removed from the wall of the longitudinal extension (2) of the pipette, before partially releasing the piston (3) compression on the top part (1) of the pipette to draw the system fluid (11) into the longitudinal extension (2) of the pipette sufficient to resuspend the magnetic particles (8) from the wall of the longitudinal extension (2) of the pipette or from an HGMS matrix (not shown) .
  • the piston (3) is controllably moved towards the non-magnetic plate (4) to expel the system fluid containing the magnetic particles into the fluid container. Mixing is effected by controllably repeatedly and sequentially partially moving the piston (3) away from and towards the non-magnetic plate (4).
  • FIG. 4d after sufficient incubation time for specific labelling to occur, the magnet (9) is placed at the wall of the longitudinal extension (2) of the pipette to collect the processed magnetic particles.
  • Complete collection of the magnetic particles (8) is effected by controllably and sequentially partially moving the piston (3) away from and towards the non-magnetic plate (4) so that the system fluid (11) containing the magnetic particles repeatedly flows past the wall or HGMS matrix adjacent to the magnet (9) .
  • the system fluid (11) is expelled from the pipette by moving piston (3) towards the non-magnetic plate (4) and the fluid container (10) containing the residual system fluid (11) is removed from the device.
  • the magnetic particles can be washed, as many times as required, by following the procedure detailed in FIG. 4a to d inc., substituting a wash solution for the system fluid.
  • the magnetic particles can be processed directly as will be described for figures 4h and 4i
  • FIG. 4e & 4f a detection fluid container (10) containing a detection fluid (12) , for example, containing an enzyme substrate or similar, is inserted into the device.
  • the magnet (9) is removed from the wall of the longitudinal extension (2) of the pipette, before partially releasing the piston (3) compression on the top part (1) of the pipette to draw the detection fluid (12) into the longitudinal extension (2) of the pipette sufficient to resuspend the magnetic particles (8) from the wall of the longitudinal extension (2) of the pipette, or via an HGMS matrix.
  • Mixing if required, is effected by controllably repeatedly and sequentially partially moving the piston (3) away from and towards the non-magnetic plate (4) .
  • FIG. 4g After sufficient incubation time the detection reaction to occur, the magnet (9) is placed at the wall of the longitudinal extension (2) of the pipette to collect the processed magnetic particles. Complete collection of the magnetic particles (8) is effected by controllably and sequentially partially moving the piston (3) away from and towards the non-magnetic plate (4) so that the reacted detection fluid (13) containing the magnetic particles repeatedly flows past the wall or HGMS matrix adjacent to the magnet (9) and are collected in the pipette.
  • FIG. 4h If the detection instrumentation is external to the device of the invention the reacted detection fluid (13) is expelled from the pipette by moving piston (3) towards the non-magnetic plate (4) and the reaction container (10) containing the reacted detection fluid (13) is removed from the device for further processing.
  • FIG. 4i If the detection instrumentation is integral to the device of the invention the reacted detection fluid
  • FIG. 5 shows an automated device of the invention.
  • This figure displays the side elevation of an automated device that can process a plurality of samples (not shown) using multiple pipette/bulb arrangements (only one of which is shown) using the processes described in detail for figures 1, 2, 3 and 4.
  • the top part (1) of the pipette is a cylindrical flexible-walled body with which the longitudinal extension part (2) of the pipette is an integral part. It is a similar plastic Pasteur type pipette described in the preferred embodiments detailed in FIGS. 1, 2 and 3.
  • a compressing device with piston (3) and a series of magnets (4) that are housed in a magnet holder (5) are directly in contact with the top part (1) of the pipette.
  • the piston (3) is connected to a motorized controller (6) to effect the required movement of the piston (3).
  • piston (3) When piston (3) is moved towards the magnets (4) housed in the magnet holder (5) the top part (1) of the pipette is compressed effecting fluid movement and magnetic capture of magnetic particles as previously described in detail for figures 1, 2 and 3.
  • the piston (3) has been designed to accommodate a removable non-magnetic plate (7) or a second magnet holder (as in 5) if a higher magnetic field is required in a given application.
  • the pipette/bulb arrangement (1 & 2) is held in position in the device using a fixed housing (8).
  • the magnet (4) can be displaced laterally relative to the top part (1) of the pipette by moving the magnet holder (5) , which is connected, to a magnet holder motorized controller (not shown) .
  • the magnet holder (5) When the magnet holder (5) is displaced laterally from the top part (1) of the pipette, it remains in contact with the top part (1) of the pipette and a non-magnetic face of the magnet holder (5) contacts the top part (1) of the pipette.
  • the magnet holder (5) is in the nonmagnetic position and the piston (3) moves towards magnet holder (5) the top part (1) of the pipette is compressed, effecting fluid movement within the body of the pipette, without magnetic separation occurring (i.e. during mixing or particle concentration) .
  • An additional magnet holder (9) is shown in the device for particle concentration (see figure 3 for details), or for processing the captured magnetic particles in conjunction with detection procedures (see figure 4 for details) .
  • This magnet holder is a duplicate of magnet holder (5) and therefore contains both magnet positions and non-magnetic positions.
  • the magnet holder is connected to the magnet holder motorized controller (not shown) and can be moved in the same plane as magnet holder (5) i.e. displaced laterally from the pipette longitudinal extension (2) to present a magnet or non-magnetic plate as required (refer to figure 3 and 4 for details) .
  • the device has a mobile platform (10) for retaining and moving the fluid containers (not shown) used in the assays (see figures 1, 2, 3 and 4 for details) .
  • This mobile platform (10) is connected to a motorized controller (not shown) to effect the vertical movement of the fluid containers with the result that the opening at the tip at the end of the longitudinal extension (2) of the pipette is inserted into the fluid of choice retained within the fluid containers as and when required.
  • An apparatus (not shown) is located on the mobile platform
  • the method according to the present invention can be applied to various types of apparatus, and in this case a mechanism required for controlling magnetic particles in a reaction can be substantially improved.
  • the magnet was removed and 1 ml of wash buffer was drawn into the pipette to resuspend the magnetic particles from the inner wall of the pipette bulb.
  • the magnet was placed towards the end of the longitudinal extension of the pipette, approximately 20 mm from the opening at the end of the longitudinal extension of the pipette and the 1 ml wash fluid was slowly (taking approximately 10 seconds) ejected from the body of the pipette.
  • the magnetic particles were concentrated at the inner wall of the longitudinal extension of the pipette adjacent to the magnet.
  • a 50 ⁇ l aliquot of 0.1 M HC1 was prepared in a microtube (Eppendorf) .
  • the magnet was removed from the pipette and the 50 ⁇ l HC1 solution was drawn into the pipette to resuspend the magnetic particles, before ejecting the acid solution containing the magnetic particles back into the microtube. Oocysts were dissociated from the magnetic particles and enumerated by microscopy using standard methods as described elsewhere (US EPA Method 1622) .

Abstract

L'invention concerne un procédé et un dispositif permettant de mélanger de manière efficace des particules magnétiques dans des fluides, de les séparer de ces fluides, de les recueillir et de laver. Le fluide dans un récipient approprié contenant les entités cibles et les particules magnétiques présentant des affinités avec lesdites entités cibles, est placé dans un tel dispositif d'une manière amovible et le mélange efficace est réalisé en sus d'un système collecteur à champ magnétique. Les particules magnétiques en volumes restreints peuvent être lavées et concentrées. L'invention concerne en outre des procédures de manipulation et de détection ultérieures des entités cibles liées aux particules magnétiques ou au fluide résiduel. Selon le mode de réalisation employé, le dispositif peut être appliqué à des volumes s'échelonnant de moins de 100 νl à plus de 100 ml.
PCT/NO2003/000261 2002-07-29 2003-07-29 Dispositif et procede de manipulation de particules magnetiques WO2004011146A1 (fr)

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AU2003248518A AU2003248518A1 (en) 2002-07-29 2003-07-29 A device and method for manipulating magnetic particles

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NO20023599 2002-07-29
NO20023599A NO20023599D0 (no) 2002-07-29 2002-07-29 Fremgangsmåte og anordning for manipulering av magnetiske partikler, spesielt for blanding, separasjon, oppsamling ogvasking

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CN103084227A (zh) * 2012-12-13 2013-05-08 宁波大学 针对磁性复合微粒悬浊液离析程序的化学实验用装置
CN105709924A (zh) * 2014-12-22 2016-06-29 株式会社岛津制作所 磁性体粒子操作用装置
EP3409365A1 (fr) * 2017-05-31 2018-12-05 Sysmex Corporation Procédé de séparation solide-liquide, appareil de séparation solide-liquide et kit destiné à être utilisé à cet effet
EP3445495A4 (fr) * 2016-04-22 2019-04-03 Purdue Research Foundation Capture et analyse de particules à haut débit
CN110455790A (zh) * 2019-08-19 2019-11-15 中国石油大学(华东) 一种啶虫脒检测装置及检测方法
US11440009B2 (en) * 2016-07-15 2022-09-13 Hewlett-Packard Development Company, L.P. Plurality of filters
WO2024048660A1 (fr) * 2022-08-31 2024-03-07 公益財団法人川崎市産業振興財団 Procédé de récupération de bille magnétique et dispositif de récupération de bille magnétique

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WO2009122216A1 (fr) * 2008-04-04 2009-10-08 Alaska Food Diagnostics Limited Système d'essai basé sur la séparation immunomagnétique pour détecter des micro-organismes dans un échantillon d'aliments
CN103084227A (zh) * 2012-12-13 2013-05-08 宁波大学 针对磁性复合微粒悬浊液离析程序的化学实验用装置
CN103084227B (zh) * 2012-12-13 2016-08-03 宁波大学 针对磁性复合微粒悬浊液离析程序的化学实验用装置
CN105709924A (zh) * 2014-12-22 2016-06-29 株式会社岛津制作所 磁性体粒子操作用装置
EP3445495A4 (fr) * 2016-04-22 2019-04-03 Purdue Research Foundation Capture et analyse de particules à haut débit
US11440009B2 (en) * 2016-07-15 2022-09-13 Hewlett-Packard Development Company, L.P. Plurality of filters
JP2018205043A (ja) * 2017-05-31 2018-12-27 シスメックス株式会社 固液分離方法、固液分離装置およびそれに用いるピペットチップ、粒子およびキット
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US10702864B2 (en) 2017-05-31 2020-07-07 Sysmex Corporation Solid-liquid separation method, solid-liquid separation apparatus, and kit to be used therefor
EP3409365A1 (fr) * 2017-05-31 2018-12-05 Sysmex Corporation Procédé de séparation solide-liquide, appareil de séparation solide-liquide et kit destiné à être utilisé à cet effet
CN110455790A (zh) * 2019-08-19 2019-11-15 中国石油大学(华东) 一种啶虫脒检测装置及检测方法
CN110455790B (zh) * 2019-08-19 2022-05-13 中国石油大学(华东) 一种啶虫脒检测装置及检测方法
WO2024048660A1 (fr) * 2022-08-31 2024-03-07 公益財団法人川崎市産業振興財団 Procédé de récupération de bille magnétique et dispositif de récupération de bille magnétique

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