WO2011027146A2 - Procédé magnétique et ultrasonore - Google Patents

Procédé magnétique et ultrasonore Download PDF

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Publication number
WO2011027146A2
WO2011027146A2 PCT/GB2010/051434 GB2010051434W WO2011027146A2 WO 2011027146 A2 WO2011027146 A2 WO 2011027146A2 GB 2010051434 W GB2010051434 W GB 2010051434W WO 2011027146 A2 WO2011027146 A2 WO 2011027146A2
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WIPO (PCT)
Prior art keywords
chamber
particles
wall
acoustic
paramagnetic particles
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WO2011027146A3 (fr
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Paul Birch
Damian Joseph Peter Bond
Christopher John Stanley
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PROKYMA TECHNOLOGIES Ltd
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PROKYMA TECHNOLOGIES Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/0009—Settling tanks making use of electricity or magnetism
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/28—Mechanical auxiliary equipment for acceleration of sedimentation, e.g. by vibrators or the like
    • B01D21/283—Settling tanks provided with vibrators
    • 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
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • 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
    • B01D—SEPARATION
    • B01D2221/00—Applications of separation devices
    • B01D2221/10—Separation devices for use in medical, pharmaceutical or laboratory applications, e.g. separating amalgam from dental treatment residues
    • 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
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • 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 or biological applications

Definitions

  • This invention relates to methods and apparatus particularly for extraction and
  • Diagnostic assays as well as cell separation techniques often utilise a ligand to isolate a specific analyte or cell through a specific marker on the cell surface.
  • the ligand is attached to a solid phase to enable the specific cell or analyte to be retained, whilst non-specific items are removed by washing them away.
  • Antibodies are one widely used example of a ligand, but others include nucleic acid probes. In some cases charge on a surface is sufficient as used in capture of released DNA on a silica particle.
  • a variety of solid phases can be employed, but particles are a popular and widely utilised format. Particles with a polystyrene outer shell are particularly favoured because their density is close to water and this allows easy mixing and because ligands and other biological capture molecules easily bind to the surface.
  • Centrifugation or filtration methods can be used with polystyrene particles but increasingly use of a magnetic field on a particle with a metal oxide core is preferred.
  • Such particles are designed with a paramagnetic core of ferrite material, with a coat of polystyrene surrounding it to produce the preferred size and provide a surface for the ligands to bind.
  • Paramagnetic cores are chosen because they have no magnetic attraction of their own and therefore particles can remain monodispersed in solution, but if a magnetic field is applied, they are attracted and drawn to the magnet.
  • the reactions are performed in a tube or micro titre well.
  • Particles are added to the sample in the tube without any magnetic field present and allowed to mix, binding any analytes or cell surface antigens in the sample.
  • a magnetic field is applied usually by bringing a solid state magnet to the side or bottom of the tube. The particles are drawn to the magnet and the supernatant can be siphoned off, usually by pipette. The magnetic field can then be removed and washing buffers added. The process is usually repeated 3-5 times to ensure efficient washing of the particles, before the next stage of the procedure is followed.
  • the particle size range is very limited.
  • polystyrene particles are favoured in diagnostic applications due to their density close to water, i.e. 1. At this density particles can be easily be kept suspended in a sample as they sediment very slowly. It also ensures that they are suspended equally throughout the volume of the liquid sample, maximising the likelihood of the ligand meeting a target to bind.
  • the rate of interaction is determined by Brownian motion as the targets are small and move through the liquid to meet the particles / ligands. The rate of interaction determines the incubation period and therefore the time for the assay or protocol. In general all methods seek to speed up the protocol time to increase throughput and minimise hands on time.
  • the density of the polystyrene particles are not affected by size and size ranges of polystyrene particles range from 10 nm to ⁇ in diameter with benefits in different applications.
  • in-vivo imaging methods prefer particles of 10-50 nm, which will be actively taken into cells by methods such as phagocytosis
  • latex agglutination methods prefer particles of 100 nm due to the even, milky background which aids visualising speckling in a positive reaction
  • membrane lateral flow applications prefer coloured particles in the range of 100-200 nm due to their ability to be carried through pores in a porous material such as nitrocellulose by capillary action and other methods prefer 10-100 ⁇ diameter particles due to their ease of separation using a filter membrane.
  • Paramagnetic particles need to achieve a combination of slow sedimentation rate to enable capture of target, with rapid attraction by a magnetic field to allow separation.
  • the metal oxides used for the paramagnetic core are dense and tend to sediment rapidly. Consequently paramagnetic particles in diagnostic or cell separation applications have a thick coating of polystyrene to counteract the density and retain particle buoyancy.
  • Ferrite is the most commonly used material for a paramagnetic core and most commercially available particles are available in a narrow size range of 1.0 to 5 ⁇ diameter.
  • Commercial cell separation products such as the DynalTM range from Invitrogen or the CaptivateTM range from LAB M have standardised at 2.7 ⁇ diameter for bacterial capture and 4.5 ⁇ for larger cells.
  • the particles are smaller than 1.0 ⁇ in diameter, the paramagnetic core is too small to allow a magnetic field to rapidly draw them to the wall of the tube, thus increasing the assay time. If they are larger than 5 ⁇ , they sediment rapidly and do not enable efficient capture of target in the bulk of the sample. Consequently paramagnetic particles have only been applied to a limited number of applications compared to non-magnetic polystyrene particles.
  • Proteins are also particularly prone to stick to surfaces such as plastics or glass which also have hydrophobic surfaces. If a paramagnetic particle coated in biological materials is drawn to the surface of a plastic tube, vigorous methods are required to re-suspend them after the magnetic field is removed. These include pipetting, vortexing and shaking. In some cases the particles do not return to their mono-dispersed state, thereby removing ligand - antigen complexes on their surface from ensuing reactions. Other particles do not leave the wall of the tube or are lost in the aspiration processes. Particle recovery levels are not widely reported, but there are clearly significant losses.
  • Antibody coated magnetic particles are widely used to capture pathogenic bacteria or in DNA purification as a step in analysis using the Polymerase Chain Reaction (PCR) or other nucleic acid amplification method. Vigorous mixing can lead to aerosol generation, with associated health risk in the case of pathogenic bacteria and a cross contamination risk in PCR analysis.
  • PCR Polymerase Chain Reaction
  • An ultrasound or acoustic standing wave field is capable of localising particles within a liquid at either the pressure nodes or antinodes of the field. Localisation is dependent upon a number of different factors including the relative densities and compressibility of the particles and the fluid.
  • An acoustic standing wave field is produced by the superimposition of two waves of the same frequency travelling in opposite directions either generated from two different sources, or from one source reflected from a solid boundary.
  • Such fields are characterized by regions of zero local pressure (acoustic pressure nodes) with spatial periodicity of half a wavelength, between which areas of maximum pressure (acoustic pressure antinodes) occur.
  • Ultrasound is sound with a frequency over 20,000 Hz. It has long been established that acoustic radiation force generated in an ultrasound standing wave resonator can bring evenly distributed particles/cells in aqueous suspension to the local pressure node or antinode planes.
  • the radiation force arises because any discontinuity in the propagating phase, for example a particle, cell, droplet or bubble, acquires a position- dependent acoustic potential energy by virtue of being in the sound field. Suspended particles tend therefore to move towards and concentrate at positions of minimum acoustic potential energy.
  • the lateral components of the radiation force which are about two orders of magnitude smaller than the axial, act within the planes and concentrate cells/particles in a monolayer. This phenomenon has successfully been used to aggregate clumps of particles or cells and to use flow of liquids to control the sequence of reactions and discriminate between positive and negative results in antibody labelling or cross linking (agglutination) reactions as described in
  • Patent Citation 0001 WO WO 2008/037993 A (PROKYMA TECHNOLGIES
  • Patent Citation 0002 WO WO 2009/118551 A (PROKYMA TECHNOLGIES
  • a method of processing of a sample mixed with paramagnetic particles includes forming an ultrasound standing wave to drive the paramagnetic particles into one or more pressure nodes and exposing the sample mixed with paramagnetic particles to a pulling force to draw the paramagnetic particles.
  • a method of processing a sample comprises the steps of
  • the pulling force pulls the paramagnetic particles towards a wall of the chamber when the strength of the acoustic standing wave is reduced or the acoustic standing wave eliminated.
  • the paramagnetic particles are washed while they are held by the pulling force at a wall of the chamber, preferably by passing a laminar fluid flow over the paramagnetic particles.
  • the standing acoustic wave is reduced or eliminated and then reestablished on a plurality of occasions before the particles are flushed from the chamber.
  • the pulling force can be gravitational it is preferred that it is exerted by a magnetic field. Applying a magnetic field to the chamber as the sample is admitted to the chamber dampens kinetic energy of the paramagnetic particles and speeds up aggregate formation.
  • a preferred alternative to cycling, reducing or eliminating the standing acoustic wave and then re-establishing it is either to increase or decrease the magnetic field on a cyclic basis while paramagnetic particles are held in an acoustic wave of constant strength.
  • a magnetic field is applied continuously to the chamber while any paramagnetic materials are present in the chamber. This has the advantage that it acts as a brake on the paramagnetic particles when they enter the chamber, reducing the propensity of the particles to be flushed from the chamber with any fluid flow.
  • sample containing biological particles particularly bacteria.
  • paramagnetic particles should have surfaces that will capture biological particles.
  • a method can be part of a process to extract biological cells from samples containing materials that can act as inhibitors for detection methods and is especially useful, for example, when the sample is one to be tested for Mycobacterium tuberculosis.
  • a particular aspect of this invention is the possible use in the method of a disposable chamber and disposing of the chamber after processing each sample.
  • the magnets may be permanent magnets, electromagnets or solid state magnets, or any other device generating a magnetic field.
  • An ultrasound standing wave is highly efficient in pushing the paramagnetic particles off the chamber surfaces and back into the nodal plane or planes. At which point, reactions can be controlled, by introducing buffers containing reagents, which actively mix and react. Positioning of transducers to generate ultrasound standing waves in chambers of different geometries have been widely described and include tubular or capillary shapes, circular, square, rectangular or hexaganal to mention a few. This description shall refer to chambers with flat walls on the base and ceiling of the chamber, but the invention is not restricted to these presentations.
  • the one or more magnets are normally positioned on the outside of a wall of said first chamber which is normally the acoustic reflecting wall of the chamber.
  • a close contact (coupling) is required between the transducer and the chamber to allow transfer of acoustic energy from the transducer into the chamber.
  • Multilayer chambers have been described where the coupling is achieved using a physical contact mechanism, such as an adhesive or a liquid coupling layer, such as glycerol or oil.
  • the wall opposite the acoustic reflecting wall comprises a flexible thin film material.
  • the acoustic transducer can be affixed to the outside surface (facing into the chamber) of the flexible thin film material.
  • the thin film material used is about 20 ⁇ thick.
  • the acoustic transer can be coupled to the chamber without a physical contact mechanism such as an adhesive or liquid coupling layer, such as glycerol or oil.
  • the apparatus is operable over a range of frequencies to generate aggregates; changes in frequency are observed to assist in the formation of aggregates.
  • One way of achieving the coupling of the transducer to the thin film material without an adhesive or liquid coupling layer is to reduce the pressure on the outside surface of said thin film material.
  • Another mechanism is to use an elastic film and to press the layer across the transducer to stretch the film and form a tight fit.
  • the paramagnetic particles can be gathered in the outlet by placing a magnet against the outlet to collect them.
  • a small second chamber can be provided in the outlet with a magnet positioned against its wall.
  • the chamber and the inlet and outlet are disposable and are mountable in a frame.
  • a chamber for use in the analysis of biological samples is characterised in that said chamber is a one piece moulding with inlet and outlets, side walls and an acoustic reflecting wall, and a thin film wall disposed opposite said acoustic reflecting wall and attached to the side wall by lamination.
  • said moulding consists of materials which can be produced at low cost by techniques such as injection moulding such as polystyrene, polycarbonate, acrylic or other polymeric plastic materials.
  • Using a thin film enables the chamber to be operated over a range of frequencies to form the aggregate.
  • the acoustic transducer attached to the outside surface of the thin film without adhesive applying a reduced pressure to the outside surface of the thin wall, by using a liquid coupling layer, reduced pressure or by plastic deformation of the thin wall;
  • a key aspect of the invention is the use of ultrasound in a chamber to resuspend paramagnetic particles that have already been captured onto a surface by one or more magnets ; the resuspension is near instantaneous and highly efficient and the process can be repeated as often as required to facilitate washing and reagent addition/removal to/from the particles.
  • the ultrasound resuspension overcomes one of the key disadvantages of magnetic separation, namely clumping of the particles in a strong magnetic field and subsequent poor resuspension. This is a highly efficient way of resuspending paramagnetic particles without introducing vigorous mechanical means such as agitation/shaking/repeated pipetting.
  • the invention has the further advantage over previous approaches for processing of paramagnetic particles to which biological moieties are attached and that is in the case of, for example, bacterial pathogens, the method provides significant operator safety as once a sample is introduced into the enclosed chamber, subsequent procedures can take place in the chamber minimizing the possibility of operator exposure to the sample.
  • the materials of interest may be biological moieties including proteins, enzymes, hormones, bacterial and eukaryotic cells; and in that case the paramagnetic particles have surfaces that have been modified to capture specific biological materials.
  • the technique can also be applied, for example, to a sample containing DNA and possibly to the extraction of materials generated in chemical reactions.
  • the method of this invention may be used as part of a process of separation of
  • sample is sputum which may contain Mycobacterium tuberculosis.
  • sample is a blood sample.
  • the paramagnetic particles may be washed while held at a pressure node.
  • apparatus for the analysis of biological samples comprises at least one chamber, an acoustic transducer capable of creating at least one pressure node in a fluid contained in the chamber, and one or more magnets associated with the chamber to attract paramagnetic materials contained in a sample.
  • An ultrasound standing wave is highly efficient in pushing the paramagnetic particles off the top or bottom chamber surfaces (where the transducer is attached to the outside of the bottom surface and the top surface is the reflector) and back into the nodal plane or planes. At which point, reactions can be controlled, by introducing buffers containing reagents, which actively mix and react.
  • the invention provides a simple way to improve the efficiency of liquid mixing with the paramagnetic particles by turning off the ultrasound standing wave and allowing the particles to pull towards a wall, with resultant deformation of the aggregate structure and then to reapply the standing wave to reform an aggregate that is held against a flow. This process can be cycled multiple times ensuring unimpeded access of liquids to the centre of the aggregate and more efficient mixing.
  • Ultrasound forces affect particles on the basis of their size and density. When using ultrasound forces to manipulate polystyrene particles, sizes of 5 ⁇ or larger are preferred. When using paramagnetic particles, the increased density allows improved performance on 1.0 ⁇ particles. This leads to the ability to use larger particle numbers and consequently a larger surface area to volume ratio, with improvements in assay performance, speed and sensitivity. Also, due to the ultrasound force lifting paramagnetic particles that would otherwise sediment; larger paramagnetic particles can be employed if required.
  • the force of the ultrasound pushing paramagnetic particles into a nodal plane can exceed the strength of a magnetic field pulling paramagnetic particles to the floor or ceiling of the chamber.
  • This has allowed the reaction to be controlled by cycling the ultrasound power on or off whilst leaving the magnetic field active.
  • This has an advantage of minimising particle loss when a flow is applied as any paramagnetic particles escaping the ultrasound field are attracted to the floor or ceiling of the chamber.
  • the capture efficiency is defined as the ability of the aggregate of paramagnetic particles to withstand flow rates without loss of paramagnetic particles.
  • the strength of the magnetic field can be controlled electronically if using an electromagnet, or by varying the proximity of a solid state magnet to the chamber wall.
  • the combination of forces has also allowed the inventors to use a larger chamber than that used in known ultrasound devices and thus increase the volume of samples processed in a short time frame as required for diagnostic tests.
  • Previous attempts to fabricate an ultrasound chamber that could hold 1 ml of sample were unsuccessful and inefficient, as this required a large ultrasound transducer that consumed power and heated the chamber to a temperature that denatures biological materials.
  • the ability to hold non-paramagnetic particles against a flow was also observed to be poor and the recoveries of the target biological materials were less than 50%.
  • Using a combination of magnetic fields with ultrasound standing waves according to the invention has allowed the fabrication of a chamber to process 1 to 10 ml of sample volume, with greater particle holding power and using less power.
  • analyte or biological moiety is particularly intended to mean a bacterial cell, blood cell, blood platelet, cell fragment, spore, plasmid or virus, but also includes synthetic particles which may or may not be modified, or coated, with one or more different chemical or biological moieties or synthetic derivatives thereof.
  • synthetic particles include, but are not limited to, polymers, such as latex and polystyrene, composites such as gold coated polystyrene, particles with a paramagnetic core and glass/silica beads that may or may not be coated with proteins, capture moieties, recognition elements, ligands, amplification moieties or other chemical or biological agents.
  • flow rates and voltages relate to flow rates and voltages used by the inventors on their apparatus.
  • the appropriate flow rates and voltages may vary depending on the exact configuration and parameters of the ultrasound system in use, and appropriate calibration will be required. This step is within normal experimental skills.
  • Figure 1 shows a side section and Figure 2 a top view of a separation apparatus
  • Figure 3 is a longitudinal section through a separation apparatus with a disposable chamber on the line B-B' of figure 4;
  • Figure 4 shows a transverse section through the separation apparatus of Figure 3.
  • the separation apparatus comprises a circular stainless steel support 11, with an internal circular lip 11a on which is mounted, from below a thin stainless steel layer 15.
  • the internal circumference of the lip and the layer 15 define the side and bottom of a chamber 14.
  • An inlet 16 and an outlet 17 are formed through the support 11 and lip 11a to the chamber 14.
  • a piezoelectric ultrasound transducer 12 is mounted below the layer 15 such that the centre of the transducer lies on the same axis as the centre of the chamber 14.
  • a glass or quartz glass reflector 13 is mounted above the chamber 14; the diameter of the reflector is greater than that of the chamber 14 so that it can be sealed to the lip 11a, and seal off the chamber.
  • the arrangement is such that the layer 15 couples the transducer 12 to the chamber 14 and the reflector 13 will allow for the creation of standing waves in any liquid in the chamber 14, when the ultrasound transducer is turned on.
  • the gap between the layer 15 and reflector 13 is a multiple of one half the
  • the transducer 12 has a nominal frequency of 1.5MHz, and a gap of 500 ⁇ across the chamber 14 between the layer 15 and the reflector 13 represents one 1 ⁇ 2 wavelength.
  • the chamber 14 would be filled through the inlet 16 by pumping from a
  • peristaltic pump 19 a sample to be mixed in a fluid comprising a standard buffer solution.
  • the sample is blood suspected of containing bacteria.
  • the ultrasound transducer 12 can be turned on forming one or more pressure nodes in the fluid.
  • a magnet 20 which may be a permanent magnet, an electromagnet or a solid state magnet.
  • Example 1 describes the evaluation of the chamber with paramagnetic particles
  • Example 2 describes the isolation of TB ⁇ Mycobacterium tuberculosis) from a 3 ml treated sputum sample and the use of the separation apparatus of Figures 1 and 2 to isolate the target TB bacteria.
  • Step 1 12.5 ⁇ 1 of Lodestar Str-A beads (2.7 ⁇ paramagnetic particles, obtainable from Polymer Laboratories, UK) were added to 87.5 ⁇ 1 of 0.01M PBS pH7.4;
  • Step 2 A permanent magnet 20 was placed below the chamber 14 underneath the transducer 12;
  • Step 3 The ultrasound was switched on at a frequency of 1.432 MHz;
  • Step 4 The sample mixed with the Lodestar beads was loaded into the ultrasound chamber at a flow rate setting of 0.75 on a Gilson Miniplus 3 peristaltic pump 19;
  • Step 5 An aggregate formed as the particles flowed into the chamber and they were continuously captured into the aggregate for 100 seconds;
  • Step 6 The liquid inlet was switched from the sample to a wash buffer consisting of 0.01M PBS pH7.4 at the entry tube 16;
  • Step 7 The PBS was flowed through the chamber 14 for 40 seconds at a setting of 0.75 on the Gilson Miniplus 3 peristaltic pump 19;
  • Step 8 The ultrasound standing wave was deactivated by switching off power to the transducer 12 and the Lodestar paramagnetic particles were drawn to the floor of the chamber by the magnet in less than 1 second. The morphology of the aggregate changed as the particles compacted and moved according to the magnetic field;
  • Step 9 Reactivating the ultrasound standing wave by resupplying power to the
  • transducer 12 resulted in the particles rising off the floor of the chamber 14 and forming a typical aggregate 18 that had a circular appearance when viewed from above;
  • Step 10 Switching the power to the signal generator on and off allowed a repeated controlled sedimentation and levitation of the particles between the compacted and elevated states.
  • Step 1 Using a commercial kit (TB-Beads) available from Microsens Medtech
  • Step 2 Paramagnetic beads functionalized with ligands that bind TB ⁇ Mycobacterium tuberculosis) as supplied in the commercial kit were mixed with 3 ml of the thinned sputum sample;
  • Step 3 The ultrasound chamber 14, capable of holding of liquid was pre-filled with buffer.
  • the ultrasound transducer was activated at a frequency of 1.49 MHz.
  • a magnet 20 was positioned under the floor of the chamber 14;
  • Step 4 3 ml of the thinned sputum sample/TB bead complex was introduced in the chamber 14 through inlet 16 in a continuous flow, and the particles along with any bound mycobacteria were observed to form into an aggregate. No particles were observed escaping at the other end through outlet 17. In this design of circular chamber 14 the aggregates 18 of particles were observed to form predominantly in one central aggregate;
  • Step 5 After 2 minutes, whilst the TB bead aggregates 18 were still in the chamber, the flow was stopped and the power of the ultrasound standing wave reduced by lowering the voltage on the transducer 12, allowing the particles to be drawn by the magnetic field created by the magnet 20 to the floor of the chamber;
  • Step 6 The chamber 14 was washed with distilled water using the peristaltic pump 19 at flow rate setting 8 with 3 ml of wash solution in 1 minute. The flow was stopped, the voltage across the transducer 12 increased to increase the power of the ultrasound standing wave and the beads were observed to return to a central aggregate in the chamber 18;
  • Step 7 The captured mycobacteria on the magnetic beads were then stained by
  • particulate material of interest, or cells can be held in one or more aggregates in an ultrasound field. Reactants or metabolites in the liquid need to encounter the particulate material to react, or be consumed, and non-specific reagents or products of cellular metabolism need to be removed to allow the process to proceed maximally.
  • the particulate materials are held in an aggregate in a chamber and a pump is used to draw liquid over and through the aggregate.
  • a pump is used to draw liquid over and through the aggregate.
  • the liquid tends to stream around the clump of particles or cells and acoustic streaming determines how much of the liquid actually reaches the centre of the particle aggregate.
  • the ultrasound force is particularly strong in pushing particulate material or cells into a nodal plane.
  • Some of the known examples use a flow perpendicular to the nodal plane to stress the aggregates formed by the ultrasound field.
  • the aggregate of particles is held less strongly in this plane and this imposes a restriction in the dimensions of the chamber that can be utilised.
  • the chambers fabricated for use with ultrasound typically have a gap between transducer (or protective carrier layer on top of the transducer) and the reflector of 1, 2 or 3 half wavelengths. In the case of a 1.5 MHz transducer this equates to a 0.5, 1 or 1.5 mm gap.
  • the liquid flow will exhibit laminar flow properties in the chamber.
  • laminar flow follows a parabolic pattern, the liquid in the centre of the chamber will have the fastest flow rate, whilst as the liquid is closer to the carrier layer or reflector, the flow will be slower until at the surfaces there is almost no flow at all. This means that the ultrasound force is holding particle aggregates at the position of maximum fluid flow, but where its resistance to movement in the flow is weakest because the strongest force is perpendicular to the nodal plane.
  • a sample under investigation is likely to contain two different types of contaminants that could interfere with analysis or reaction.
  • the first is large particles, such as non- desired cells, particularly blood cells.
  • the second is soluble factors that could bind to the particles or otherwise adversely affect the chance of interaction between a ligand and a target molecule.
  • Ultrasound is very effective at addressing the second issue. Without it, the particles alone will still rely upon the relatively slow Brownian motion in the liquid to ensure that ligands encounter their target molecule. Ultrasound stimulates acoustic streaming along at least two axes in a three dimensional space leading to a highly efficient mixing reaction and greatly speeding up the ligand - analyte interaction. This observation has previously been reported in the ability of ultrasound to speed up the reaction and also improve the sensitivity of a latex agglutination slide test for meningitis.
  • ultrasound alone is not very good at addressing the first issue, namely removing large contaminating particles or cells from the target particles as the aggregates form on the basis of size and there is insufficient discrimination between cell sizes to be effective.
  • particles move into aggregates they can also 'herd' or co-entrap smaller particles that would normally not be affected by the ultrasound.
  • Use of magnetism as described in this invention is very good at pulling paramagnetic particles to the floor or ceiling of the chamber. Since the particles are then located in the slowest part of the parabolic flow in the laminar flow in the chamber the particles can be washed with a high flow rate, thereby rapidly removing the contaminating cells.
  • the paramagnetic particles at the floor or ceiling of a chamber can prove resistant to resuspension, as reported earlier.
  • the particles will still be contaminated with soluble factors from the sample but washing is ineffective as they are in the slowest flow of the chamber.
  • By reintroducing an ultrasound standing wave in the chamber the paramagnetic particles are resuspended, and in moving between the floor and the pressure node they mix with the liquid in the chamber, thereby aiding washing. Further they may be washed while held in the pressure node and a continuing magnetic field applied to the chamber makes the paramagnetic particles resistant to being flushed from the chamber by the washing fluid.
  • chamber is used to describe any vessel that has an ultrasound transducer associated with it. It may be a chamber as shown in Figure 1, or a straight conduit with no change in diameter between the inlet and outlet, or any other vessel in which the method described may be carried out.
  • paramagnetic beads/particles are a compromise between achieving density close to 1.0 and as small a size as possible for mixing and rapid reaction kinetics.
  • particle sizes of 2.7 ⁇ do not contain sufficient paramagnetic material in the core to respond efficiently to an electromagnet.
  • the aggregation of particles into clumps provides a larger mass of magnetic material, which has been found to respond well to an electromagnet.
  • transducer generating the ultrasound standing wave can be driven at a lower voltage in the design described in this invention, a larger volume chamber can be designed without the problems of overheating reported previously.
  • a chamber was constructed with a fill volume of 0.75 ml.
  • a transducer with resonant frequency of 1 MHz (Ferroperm, Denmark) was scored on the top electrode to match the working area of the chamber and fixed to the base of the chamber using conductive epoxy resin.
  • the active transducer as connected to the chamber was 2.5 cm x 2.5 cm.
  • the chamber was milled out of Macor machinable ceramic with the base in contact with the transducer having a thickness of 0.3 mm.
  • the internal height of the chamber was 1.2 mm and the reflector layer was a soda glass double width microscope slide of 1 mm thick.
  • the larger transducer and the three nodal plane format created hundreds of aggregates of particles in three vertical layers.
  • An electromagnet outside the floor or ceiling of the chamber and driven at an amplitude of 150 and a frequency of lMHz could draw aggregates to the floor or the ceiling of the chamber once the ultrasound amplitude was decreased. It was also observed that if the aggregates were drawn to the floor of the chamber, they would settle and spread out slightly and upon increasing the amplitude of the ultrasound the particles at the centre would re-suspend, but particles that had spread out were less affected.
  • the magnet was therefore applied from above to draw the particles to the ceiling of the chamber, where gravity in addition to the ultrasound could be employed to re-suspend particles into the nodal planes.
  • Step 2 Fill chamber with bead solution
  • Step 3 Wait 2 to 3 mins for agglomeration to occur (using Lodestar 2.7um beads);
  • Step 4 Switch USW off to allow all agglomerates to settle to the bottom ( ⁇ 10s);
  • Step 5 USW on to re- agglomerate on the bottom nodal plane
  • Step 6 Switch on magnet (mounted above the glass) to collect agglomerates (Magnet
  • Step 7 Flow in next chamber-full of bead solution
  • Step 8 Repeat steps 3-6 as necessary;
  • Step 10 Pump out beads (or alternatively a 2nd magnet was used to capture the paramagnetic beads near the exit port).
  • a disposable separation apparatus chamber is used.
  • Ultrasound chambers designed to agglomerate particles are typically fabricated with a rigid connection between the transducer and the floor of the chamber. Such chambers are usually fabricated out of rigid materials such as steel or ceramics such as Macor. Research has shown that sound energy propagates through rigid materials with lower losses than through flexible materials such as plastics.
  • the separation apparatus of this invention was specifically directed for use in connection with diagnostic processes, such as detection of Tuberculosis and other disease-causing organisms. Tests for these applications need to be safe, but also disposable to ensure no cross contamination between samples.
  • the manufacturing prices for the transducers and rigid chambers are too high compared to the prices paid for disposable tests and a key purpose of the invention is to allow for the separation apparatus to be fabricated out of low cost polymers (e.g. injection moulding) which requires a different mechanism of coupling to the transducer (the reusable component).
  • a disposable chamber is provided to be used as part of an instrument utilising the method of this invention.
  • the interplay of the magnetic and ultrasound fields creates a holding power stronger than either alone. Consequently it has been found that the acoustic chamber can still function to effect separation of particles with less efficient transfer of acoustic energy into the fluid in the chamber.
  • the floor of the chamber is designed to minimise losses in the transfer of acoustic energy and laminate films have proved to be usable.
  • Machine oils, glycerol or other liquids are suitable.
  • the second method illustrated in Figures 3 and 4 uses low pressure between the transducer and the base of the chamber, which was achieved by creating holes through the transducer and applying a pump to withdraw any air between the two faces.
  • the transducer can simply pushed on to the plastic and held in place by deformation of the plastic.
  • the separation apparatus 50 comprises a first chamber 52, reflector wall 53, side wall 59 and a thin film cover 62 together with an inlet 54 and outlet 56.
  • the outlet 56 has a small collection chamber 58 formed therein.
  • Inlet 54 and outlet 56 have shaped ends 55 and 57 respectively to which external tubing from other apparatus may be attached.
  • the chamber 52 with reflector wall 53, inlet 54 and outlet 56 are a single transparent acrylic moulding with an open aperture closed by the film 62, these items form a single disposable unit.
  • Chamber 52 operates with a transducer 60 with resonant frequency of 1 MHz
  • the transducer was scored on the top electrode to match the working area of the chamber (2.5 x 2.5 cm) and coupled to the film 62 using a drop of glycerol, or alternatively using a partial vacuum created by applying a vacuum pump V (not shown) through housing 64 (the transducer is not part of the disposal apparatus and is reusable). To aid coupling holes 66 are formed in the transducer 60.
  • the main body of the chamber 52 was milled from acrylic material and the open top covered by the sheet of thermal shrink film 62. This film had a thickness of 20 ⁇ .
  • the internal height of the chamber was 1.05 mm and the reflector layer was 0.35 mm thick acrylic. These dimensions produced a chamber with three half-wavelengths and therefore three nodal planes were present in the chamber when driven at a frequency of 1.740 MHz.
  • the chamber and inlet and outlet are supported in an acrylic support 63 comprising upper member 64 and lower member 68, upper member 64 pivotally mounted on a pivot 74 on lower member 68.
  • the lower member 68 has supporting legs 71 either ends to be received on a stand 70.
  • the upper and lower members 64 and 67 have moulded surfaces to receive the chamber moulding and to support it in position in the support 63.
  • Upper member 64 has a recess 65 which is connectable to a vacuum pump V (not shown), to reduce the pressure within the recess 65. When this occurs thin film 62 clings tightly to the transducer 60, thus holding it in place above the chamber 52.
  • the chamber's side walls 59 support the chamber in a recess 67 formed in lower member 64.
  • the recess 67 has an indentation 69 below the reflector wall of the chamber 53 creating an air gap below the base of the chamber to reduce transmission of vibrations form the chamber to member 68 when the transducer 60 is operating.
  • the separation apparatus 50 is mounted on a stand 70 having a cavity 72 in which control apparatus is conveniently mounted.
  • One or more magnets 76 are mounted beneath the lower member 68.
  • the magnets may be permanent magnets, electromagnets or solid state magnets, the latter of the kind which when orientated in one direction produce a magnetic field and when turned by 90° have no field.
  • the position of the magnet is controlled by a step motor 80.
  • the magnet can be placed in various positions below the chamber 52, progressively moving from right to left in Figure 4 to a final position below the collection chamber 58. In the final position the magnet is used to collect the paramagnetic particles from the chamber after the acoustic transducers have been turned off before all the particles are flushed from the chamber out of outlet 56 for further analysis.
  • transducer 60 mounted on a thin film above the chamber 52, with the magnet 76 below, the chamber has also been designed to be the other way up, with the transducer 60 below the chamber and thin film 62 forming the lower wall of the chamber.
  • the moulded shapes of members 64 and 68 would need to be different in order to accommodate this different orientation with features of the lower member 68 above moulded in the upper member and vice versa.

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Abstract

L'invention concerne un procédé permettant de traiter un échantillon mélangé avec des particules paramagnétiques, lequel procédé consiste à exposer ledit échantillon à un champ magnétique afin d'attirer les particules paramagnétiques, puis à former une onde stationnaire ultrasonore afin d'acheminer les particules paramagnétiques dans un ou plusieurs noeuds de pression. L'invention concerne également un appareil permettant d'analyser les échantillons biologiques, lequel appareil comprend une chambre, un transducteur acoustique capable de créer au moins un noeud de pression dans un fluide contenu dans la chambre et un ou plusieurs aimants associés à la chambre afin d'attirer les matériaux paramagnétiques contenus dans un échantillon.
PCT/GB2010/051434 2009-09-01 2010-09-01 Procédé magnétique et ultrasonore Ceased WO2011027146A2 (fr)

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GB0915072A GB0915072D0 (en) 2009-09-01 2009-09-01 Ultrasound & magnetic method

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