Classifications

• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
  • H01F10/00—Thin magnetic films, e.g. of one-domain structure
  • H01F10/26—Thin magnetic films, e.g. of one-domain structure characterised by the substrate or intermediate layers
  • H01F10/265—Magnetic multilayers non exchange-coupled
• • 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
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
  • H01F41/00—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties
  • H01F41/32—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties for applying conductive, insulating or magnetic material on a magnetic film, specially adapted for a thin magnetic film
  • H01F41/34—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties for applying conductive, insulating or magnetic material on a magnetic film, specially adapted for a thin magnetic film in patterns, e.g. by lithography

Definitions

• the invention relates to a magnetic particle, and methods of making and using magnetic particles.
• SPIONs superparamagnetic iron-oxide nanoparticles
• a net-zero-magnetization remanent state means that in the absence of a magnetic field, the magnetic particles have no net magnetic moment and no external stray field.
• magnetic particles are typically suspended in a liquid or fluid medium and are free to move within that medium.
• the particles’ stray fields may interact and cause the particles to agglomerate, or to clump together. This is undesirable because the purpose of using magnetic particles in biotechnology applications is to be able to steer, or direct the motion of, particles suspended in a liquid or fluid medium by applying an external magnetic field. If the magnetic particles agglomerate, then this cannot be achieved.
• the particles with zero net magnetization remanent state should also have a low susceptibility at small fields.
• particles with a high susceptibility are used, then after an applied field has been applied to direct or move the particles in a desired manner, then particles which have agglomerated during the application of the field stay agglomerated once the applied field is removed. It is understood by the skilled person that this should also be avoided in magnetic particles for biotechnology applications.
• An important biotechnology application is to carry out multiplexed immunoassays of biological samples.
• the accurate quantification of proteins in a biological sample is of significant importance for both research and clinical diagnostic applications.
• a multiplexed immunoassay simultaneously quantifies a plurality of different proteins in a given sample. Analysing protein fingerprints of samples in this way has the potential to accelerate research and to enable improved diagnostics.
• multiplexed assay systems such as Luminex (RTM), Firefly (RTM) and Fireplex (RTM) have been developed. These systems use individual particle sets in which each particle is coated with a capture antibody qualified for one specific analyte.
• the Luminex (RTM) system is based on polystyrene or paramagnetic microspheres, or beads, that are internally dyed with red and infrared fluorophores of differing intensities to allow the differentiation of one set of beads from another.
• the Firefly (RTM) and Fireplex (RTM) systems also use fluorophores to allow differentiation of one particle set from another, but in this case the particles are in the form of rods coded by applying a different fluorophore at each end. Measurement of the fluorophores again aims to distinguish one rod from another.
• these systems suffer from limited multiplexing (limited number of different proteins that can be identified) due to limited ability to distinguish with certainty between the channels of the multiplex in assay results.
• the invention may thus provide a magnetic particle, comprising a layered structure between a top surface of the particle and an opposed bottom surface of the particle, the layer or layers including one or more magnetized layers.
• the ratio of a lateral dimension of the one or more magnetized layers to the thickness, or aggregate or effective thickness, of the magnetized layer or layers is greater than 500.
• the aspect ratio of a cross section of the magnetized layer or layers may be more than 500.
• the ratio may be higher, for example being greater than 800 or greater than 1000 or 1500 or 2000.
• the particle may further comprise a non-magnetic layer, which may advantageously provide mechanical support to the magnetic layer, and may determine physical
• characteristics of the particle such as its mechanical properties and its density.
• the particle may comprise one magnetized layer, or it may comprise more than one such layer. If it comprises two or more magnetized layers, then layers may be adjacent to each other, or in contact with each other, or they may be spaced from each other with non magnetic material in between.
• the aggregate thickness, or total thickness, of the magnetically-remanent layers in a particle having more than one magnetized layer may be the sum of the thicknesses of those magnetic layers, not including any non-magnetic layers in between them.
• the layered structure may comprise many magnetized layers and/or many layers of a non-magnetic material.
• a magnetized, or magnetic, layer may for example comprise any suitable magnetic material or materials, such as a ferromagnetic material, element or alloy, or a composite of superparamagnetic nanoparticles.
• the particle is preferably substantially flat in shape, comprising one or more substantially flat layers of magnetic and/or non-magnetic materials stacked on top of one another.
• the layers are preferably of substantially the same shape and size as each other, each having the same lateral shape and size as the particle itself. However, the shape and structure of the particle may vary from this as described further below.
• the particle may have a zero or a non-zero magnetic remanence.
• the shape and structure of a particle embodying the invention displays an unexpectedly-low stray field at the surfaces of the particle, so that a plurality of the particles suspended in a fluid or liquid medium may advantageously not aggregate or clump.
• the inventors have found that this is the case whether or not the particles are fully magnetically remanent.
• the particle when an external field is applied the particle has a sufficient magnetic moment for the external field to apply a desired force to the particle.
• the desired force may depend on the application of the particle (such as in a bioassay with the particle suspended in a fluid medium).
• the applied external field is typically less than 2T, or 1T or 0.5T, and may typically be greater than 0.05T or 0.1T or 0.25T.
• the external field may be used to steer the particle within a fluid medium.
• the magnetic moment may be due to magnetization of the particle itself, or it may be induced in the particle by the external field. It is important, however, for the particle to contain sufficient magnetic material to enable the desired force to be generated.
• the magnetic moment that can be generated by an external field applied to a particle may depend on the total volume V of magnetic material in the particle multiplied by that material’s magnetization M s .
• V.M S for a particle embodying the invention is preferably greater than a predetermined value, such as 10 -18 J/T or 5x10 -18 J/T or 10- 17 J/T.
• the inventors have found that the physical distribution of the magnetic material within the particle may determine the stray field near the particle, and therefore the tendency of particles to interact with each other and/or to agglomerate.
• the inventors have found that distributing the magnetic material in the form of a layer or layers (preferably parallel layers) having a cross section with high aspect ratio AR may generate an advantageously low stray field.
• This preferred particle geometry may advantageously provide particles with low stray field and little or no tendency to agglomerate.
• the inventors have determined that the parameter AR/M S is preferably greater than 1x10 3 nrVA 1 or 3x10 3 nr 1 /A 1 or 5x10 3 nrVA 1 .
• stray field of less than about 2500 A/m (30Oe) at 10 times the layer thickness above or below the layer.
• this level of stray field may advantageously prevent agglomeration.
• AR may be a lateral dimension of a cross section of the structure divided by a thickness of the structure.
• the lateral dimension may be the minimum lateral dimension of the layer or, if the shape of the layer is more complex, then it may be preferable to consider an average lateral dimension of the layer. If the thickness of the layer is constant, then that thickness can be used for the calculation of AR. If the thickness of the layer varies, then an average thickness can be used.
• AR may be evaluated using the particle thickness.
• an alternative approach to evaluating the thickness for calculating AR may be to calculate a diluted thickness for the magnetized material. If for example, two or more parallel layers of magnetized material of aggregate thickness Tm are separated by layers of non-magnetic material of aggregate thickness Tnm , then the diluted thickness of the magnetized material would be Tm/(Tm+Tnm) .
• AR and Ms for the calculation of AR/M S 2 or AR/M S
• the effective M s may be found using the total particle volume and the total moment per particle.
• the particle or metallic layer(s) lateral dimension and thickness may be measured directly, for example using microscopy and/or electron microscopy techniques, to evaluate AR.
• Particles embodying the invention are preferably planar in shape, with their length and width both being greater than their thickness.
• the length and width of the particle, or two lateral dimensions of the particle measured perpendicular to each other are similar to each other, or differ from each other by less than about 10%, 30%, 50% or 70%.
• a particle might typically be in the form of a circular or elliptical or polygonal disc, or a generally flat cuboid with a square or rectangular perimeter.
• the top and bottom surfaces of the particle may be separated by a particle thickness of between 5 nm, or 10nm or 50nm or 100nm, and 100pm or 50pm or 5pm or 1 pm or 500nm.
• a minimum lateral dimension of the particle may be greater than 1 pm, and preferably greater than 5pm or 10pm, and a maximum lateral dimension may be less than 500pm or 200pm or 100pm or 50pm.
• a ratio of the minimum lateral dimension of the particle to the thickness of the particle may be greater than 10 or 20 or 50 and/or less than 2000 or 1000 or 500.
• those layers are preferably substantially parallel to each other. In embodiments comprising more than one magnetized layer, those layers preferably have similar shapes and/or areas as each other, and may conveniently overlap with each other, optionally completely overlapping with each other.
• the opposed top and bottom surfaces of the particle are advantageously flat, but one or both surfaces may optionally be curved or not flat without affecting the desired property of the particle having a sufficiently small stray field to avoid aggregation.
• the particle itself may thus be flat or curved. But in each case, the opposed top and bottom surfaces may advantageously be of sufficiently large area to enable features such as readable
• the shape of the particle may thus be in the form of a thin (low thickness) laterally-extended shape, such as a high-aspect-ratio cuboid or disc.
• Aspect ratio refers to the ratio of the minimum lateral dimension, or of an average lateral dimension, to the thickness.
• the magnetic particle may be described as being cylindrical in shape, the thickness of the particle being in an axial direction of the cylinder, with the peripheral shape of the cylinder preferably being selected so that it typically has an edge or edges which are convex or straight, advantageously with no re entrant corners.
• Preferred peripheral shapes are rectangular or square or circular.
• embodiments of the invention may include curved or non-flat particles, or particles with curved or non-flat upper and lower surfaces, while achieving the object of providing non aggregating magnetic particles.
• the minimum and maximum lateral dimensions of the magnetic particle differ by less than 90% or less than 70%.
• the minimum lateral dimension of the particle is greater than 5 pm, and preferably greater than 10 pm, and/or the maximum lateral dimension of the particle is less than 500 pm, and preferably less than 200 pm, 100pm or 75pm. These dimensions may be selected by the skilled person depending on the application for which the particles are being used, and requirements such as the desired mechanical strength of the particles.
• the layered structure of the magnetic particle advantageously comprises a magnetized layer and a non-magnetic layer.
• the non-magnetic layer may provide mechanical strength to the particle, and may provide a suitable substrate for the magnetized layer.
• the non magnetic layer may thus advantageously comprise a material selected from Al, Ta, Pt, Pd, Ru, Au, Cu, W, MgO, Cr, Ti, Si, Ir, Si0 , SiO, Sn, Ag, polymers, plastics, alloys of these materials, and composites or mixtures comprising these materials.
• the magnetized layer may for example comprise a magnetic multilayer stack of alternating layers of a magnetic material and a noble metal (such as Pt/CoFeB) where the pair is known to provide perpendicular magnetic anisotropy.
• a noble metal such as Pt/CoFeB
• the magnetized, layer is preferably an out-of-plane magnetized layer, but may be a differently magnetized layer such as an in-plane magnetized layer.
• a high saturated magnetic moment is desirable for the magnetic particles, in order to achieve a rapid response to an external field.
• the magnetic material is selected to achieve this.
• the layered structure of the magnetic particle may comprise more than one layer of non magnetic material, and/or may contain more than one layer of magnetized material.
• the particle may comprise a magnetized layer positioned between two layers of non-magnetic material.
• the magnetized layer may for example be a thin-film multilayer.
• the net magnetic field (the stray field) averaged across the lateral surface at or within a small distance of a top or bottom surface of the particle may preferably be less than 2500 A/m (30Oe)and particularly preferably less than 800 A/m (10Oe) or 400 A/m (50e).
• This field may be measured at the surface, or at a small distance such as 10nm, 50nm or 100nm from the surface, for example by using a magnetic atomic force microscope.
• the inventors’ experiments have indicated that these external, or stray, magnetic fields are sufficiently small to avoid agglomeration of magnetic particles.
• Magnetic particles embodying the invention may conveniently be manufactured or fabricated by lithographic processes.
• a second aspect of the invention may advantageously provide a magnetic particle having dimensions as described above, but preferably in which a top surface of the particle and an opposed bottom surface of the particle are separated by a particle thickness of between 5 nm and 200pm, a minimum lateral dimension of the particle is greater than 1 pm, and the ratio of the minimum lateral dimension to the thickness is greater than 10, and in which the particle comprises a layered structure through its thickness, the layers including one or more magnetically-remanent, or magnetized, layer(s) and one or more layer(s) of a non magnetic material.
• Such a particle may conveniently be fabricated by a lithographic process, and may comprise one or more of the features of the first aspect of the invention described herein.
• a top or bottom surface of the particle may carry readable information, such as a readable code.
• a readable code This may be for example a barcode or 2D code.
• the magnetic properties of the particle enable a suitable external magnetic field to be applied to steer or move or drive the particle through a fluid medium to a predetermined location for reading the code or information.
• a particle having a high-aspect-ratio shape with a large top or bottom surface on which a code or information is carried may be directed so that it is in contact with a substrate or other supporting surface for convenient reading of the code or information.
• a top and/or bottom surface of the particle may be functionalised, for example biofunctionalized or chemically functionalised. This may advantageously be in combination with applying readable information to the particle.
• a top or bottom surface of the particle may carry a readable code and the same or an opposite surface may be functionalised.
• a plurality of particles may be provided in which each particle carries readable information corresponding to the functionalisation of that particle.
• Such a particle may enable the performance of an assay, such as a bioassay, by providing the particle to a liquid or fluid assay sample and allowing the functionality of the particle to interact with the assay sample, for example with biological molecules or other components of the assay sample.
• a magnetic field may be applied to steer the particle to a reading position, and an assay result obtained by reading the readable code and measuring the interaction of the particle’s functionality with the assay sample.
• the identification of particles using readable information in this way may advantageously provide a multiplexed platform in which the particles can be accurately distinguished from each other.
• the use of barcodes, or 2D codes may provide a significantly more robust process for identifying different particles than in existing multiplexed assay platforms, with minimum crosstalk between plex channels.
• the use of readable information in this way may enable the use of very much larger numbers of multiplex channels than is currently possible. For example, barcoding or 2D codes may enable 1000 plex, or 10,000 plex, or more if desired.
• the invention may thus relate to lithographically defined, perpendicularly (or out-of-plane) magnetized particles, advantageously in the form of ferromagnetic microdiscs (microparticles, nanoparticles, microcarriers etc.) for use in biotechnology applications.
• these particles may be ferromagnetic microdiscs (microparticles, nanoparticles, microcarriers etc.) for use in biotechnology applications.
• these particles may be ferromagnetic
• the resultant high planar aspect ratio, ultra-thin discs, or microdiscs (which may be referred to as magnetic carriers (MCs) because of their ability to carry functionalisations such as biofunctional antibodies for diagnostic tools) are ferromagnetic with high magnetic moment.
• the MCs may be lithographically-defined.
• the MCs do not agglomerate when suspended in a fluid because the aspect ratio of each magnetic layer (typically 1 nm, or 5nm, total magnetic layer thickness and tens of pm in lateral size), and magnetization direction perpendicular to the plane of the MC results in a negligible stray magnetic field from each particle.
• the MCs may be characterized by a magnetization direction parallel to the surface normal of the microdisc, as well as coercive magnetization reversal, and a high magnetic anisotropy. These properties may all enable a higher degree of control over their magnetic response and hence their mechanical behaviour in a fluid under the influence of an external magnetic field.
• the physical vapour deposition process which is preferably used to fabricate the particles, or MCs, enables sub-nm control in the deposition of the magnetic thin films that form the MCs, and thus offers extreme precision in the engineering in the magnetic properties of the MCs. This may advantageously enable them to be tailored to different applications.
• barcodes (or other readable information) may be lithographically added to the surface of MCs, and the surface materials may be chosen for optimal functionalization with molecules of interest.
• FIG 1 illustrates steps in two processes, Process A and Process B, for the fabrication of magnetic particles according to first and second embodiments of the invention
• Figure 2 is a polar magneto-optical Kerr effect (MOKE) measurement of the magnetic response of the magnetic thin film Au(100.0)/Ta(2)/Pt(4)/CoFeB(0.6)/Pt(1.2)/
• MOKE polar magneto-optical Kerr effect
• Figures 3(a) and 3(b) illustrate how the readable code and the magnetic states in particles according to the embodiments are linked to ensure that the readable code may always be aligned to an external detector, such as a camera or barcode reader, by an applied magnetic field, and show images of readable codes of particles imaged by a detector;
• an external detector such as a camera or barcode reader
• Figure 4 illustrates stray field strength as a function of distance from the surface of a particle manufactured according to the embodiments.
• Figures 5(a) and 5(b) illustrate a functionalised particle according to a further embodiment of the invention, suitable for a bioassay, and illustrate the use of particles according to the further embodiment of the invention to implement a streamlined multiplex assay.
• a specific embodiment of the invention involves the fabrication of high magnetic moment microparticles made from ultrathin perpendicularly-magnetized CoFeB/Pt layers.
• the high aspect ratio of the shape of these particles results in an extremely low stray magnetic field from each particle, such that the magnetic nanoparticles show no inter-particle interaction (and therefore no agglomeration).
• an external magnetic field is applied, the particles transition to magnetic saturation with coercive, sharp switching and are fully remanent.
• Individual barcodes are added to the particles using a simple and robust lithography process and can be read optically.
• a robust multiplexed assay for example a cytokine assay, using the magnetic particles has been demonstrated highlighting their potential in assay applications.
• lithographically fabricated magnetic particles may advantageously achieve high magnetic moment, no interparticle interaction, a large surface area for functionalization, and robust particle specific barcoding. These particles may be referred to as magnetic carriers (MCs) in view of their ability to carry both functionalization and readable information.
• MCs magnetic carriers
• the large surface area of the particles may advantageously provide more area for functionalisation than in conventional assay particles.
• Lithographically defined magnetic nanoparticles are known in the prior art, for example in T. Vemulkar, R. Mansell, D. C. M. C. Petit, R. P. Cowburn, and M. S. Lesniak,“Highly tunable perpendicularly magnetized synthetic antiferromagnets for biotechnology applications,” Appl. Phys. Lett., 2015, in H. Joisten et al.,“Self-polarization phenomenon and control of dispersion of synthetic antiferromagnetic nanoparticles for biological applications,” Appl. Phys. Lett., vol. 97, no. 25, p. 2531 12, 2010, and in S.
• the MCs used here do not require the engineering of a net zero remanent magnetization state to prevent particle agglomeration.
• the MCs used here may optionally have net-zero remanence (and susceptibility to the generation of a magnetic moment in an external field) but despite the conventional expectation of the skilled person, they do not require net-zero remanence to avoid agglomeration.
• the stray field of the particles is sufficiently low to avoid agglomeration due to the shape of the magnetized material in the particle, and/or the shape of the particle, whether or not the remanent magnetization in the absence of an external field is zero.
• the MCs in the embodiment are extremely high aspect ratio cuboids, with planar length and width of 40 microns, and thickness of approximately 150 nanometres.
• Process A is illustrated in Figures 1.A1 to 1 A1 1 .
• a sacrificial layer 2 of 50 nm of Al is grown by magnetron sputtering on a Si substrate 4.
• the base 6 of the particle thin-film stack is then grown on top of this sacrificial layer, also by magnetron sputtering.
• This base consists of the following 1 1 layers (thickness in nm):
• the photoresist is then removed in a solvent such as acetone, and a new layer of photoresist 18 is spin-coated on top of the particle base 6 and the barcodes 16 as shown in Figure 1 .A6.
• a solvent such as acetone
• photoresist 18 is spin-coated on top of the particle base 6 and the barcodes 16 as shown in Figure 1 .A6.
• This is exposed in a second lithographic patterning process using a mask 20 to define the shape of the particles.
• the plurality of holes 22 that define the particle shape are aligned such that the barcodes are aligned at the centres of the holes.
• the thickness of the Au is however sufficiently thin to allow the barcode to be read through the Au layer.
• the photoresist 18 is then removed in a solvent such as acetone, and then the entire sample is subjected to ion-beam milling 28, a standard subtractive patterning process. Any thin film not protected by the ion-beam-milling hard mask is milled away. Thus, the milling removes all of the thin film that forms the base of the particle thin film stack that is not within the defined particle shapes. The milling process is stopped when the sacrificial layer is reached. Any remaining Al hard mask 26 may be removed by dissolution in a 10-30 min soak in 3-5% tetramethylammonium hydroxide solution, or equivalent Al solution etchant. Thus, photolithography patterning determines the planar shape of the particles, and the physical vapour deposition process determines their thickness and composition.
• a solvent such as acetone
• the particles 30 with barcodes, the MCs are fully defined and lie on top of the sacrificial layer.
• a magnetic field 32 greater than the coercive field for the magnetic thin film of the particles is then applied to ensure that all of the particles are magnetised out-of- plane, in an“up” state, perpendicular to the top and bottom surfaces of the particles, as shown in Figure 1.A10.
• the particles may all be magnetised in a“down” state. This links the magnetization of the particles to the physical structure of the particles in the vertical direction, allowing for alignment of the barcodes in any downstream steps such as re-deposition as shown in Figure 3 or analysis in solution.
• the sacrificial layer of Al beneath the particles is dissolved in an appropriate solvent to lift the particles 30 off the substrate and release them into solution in a fluid medium.
• Process B is illustrated in Figure 1.B1 to 1 B1 1 .
• a photoresist layer 50 is spin-coated over a Si substrate 4. It is then exposed in Figure 1.B2 using a photomask 52 to create a plurality of islands or pillars of photoresist 54, on which a series of layers of material 56 are deposited in Figure 1.B3 using magnetron sputtering to form the base 58 of the layered, thin-film structure of the magnetic particles.
• the shape of the islands or pillars defines the shape of the particles.
• Figure 1.B3 thus illustrates the structure after the deposition of the first layers of the particle.
• a lithographically-defined barcode is then added to the particles.
• a second layer of photoresist 60 is applied, and is exposed as shown in Figure 1.B5 using a photomask 62 patterned with the desired barcode for each particle.
• the photoresist is developed, and then flood-exposed 64 to allow for its removal in developer downstream.
• the bottom layer of each photoresist island or pillar 54 is shielded from this exposure step by the presence of the particles on top of the islands.
• a barcode contrast material 66 such as 15 nm of Ta is grown on top of the particles in Figure 1 .B8.
• the top layer of photoresist is then completely removed using developer in Figure 1 .B9 and the particle cap 68 is deposited consisting of 30-40 nm of Au.
• the bottom layer of resist remains intact.
• the thickness of gold is selected to ensure complete coating of the MCs (on both top and bottom surfaces) with Au for biocompatibility and to provide a surface for biofunctionalization.
• the thickness of the Au is however sufficiently thin to allow the barcode to be read through the Au layer.
• photolithography patterning determines the planar shape of the particles, and the physical vapour deposition process determines their thickness and composition.
• the particles with barcodes, the MCs, 70 are fully defined and lie on top of the islands of photoresist.
• a magnetic field 72 greater than the coercive field for the magnetic thin film of the particles is then applied to ensure that all of the particles are magnetised out-of-plane, in an“up” state, perpendicular to the top and bottom surfaces of the particles, as shown in Figure 1.B10.
• the particles may all be magnetised in a“down” state. This links the magnetization of the particles to the physical structure of the particles in the vertical direction, allowing for alignment of the barcodes in any downstream steps such as re-deposition as shown in Figure 3 or analysis in solution.
• the thin film structure of the MCs described in this embodiment in Processes A and B is thus defined as a base of (thicknesses in nm): Au(100.0)/Ta(2)/Pt(4)/CoFeB(0.6)/Pt(1.2)/ CoFeB(0.6)/Pt(1.2)/CoFeB(0.6)/Pt(1.2)1 CoFeB(0.6)/Pt(5.0).
• a 15 nm Ta barcode is on top of this layer, and this is then capped with 30-40 nm of Au.
• the thinner Au at the top face allows for imaging the barcode through the Au, and thus the barcode is only visible through the top face of the particle in the embodiment described here.
• linking the particle magnetization to the physical structure of the particle at this juncture is necessary to enable control and orientation of the barcoded face of the particle once in solution.
• Figure 2 is a polar magneto-optical Kerr effect (MOKE) measurement of the magnetic response of the magnetic thin film Au(100.0)/Ta(2)/Pt(4)/CoFeB(0.6)/Pt(1.2)/
• MOKE polar magneto-optical Kerr effect
• the thin film magnetization is clearly out of plane with sharp, coercive magnetic switches to saturation. He denotes the coercive field, or the field required to magnetically switch the thin film to its saturated magnetic state.
• Figure 3a shows how the barcode (or other 2D code) and the magnetic states of the particles 30, 70 are linked to ensure that the code may always be aligned to an external detector.
• a field above Fl c is used to set the magnetization of the particles in the“up” state before the particles are lifted off into solution.
• Figure 3b shows images of particles on a planar substrate and displaying readable codes such as barcodes.
• Figure 4 illustrates the stray field strength as a function of distance from the surface of a particle 30, 70 of the embodiments. It can be seen that the stray field is low, due to the high-aspect-ratio geometry of the particle. This advantageously reduces any tendency for the particles to agglomerate.
• Particles according to an embodiment of the invention may be used to implement a multiplex assay as follows. Steps in the process are illustrated in Figure 5 (a) and (b).
• Analyte detection is performed with a conventional sandwich immunoassay.
• the capturing antibody captures a target protein 106
• exposure of the magnetic particle to a fluorescently-labelled detection antibody 108 complementary to the capturing antibody binds to and labels the protein.
• fluorescence of the fluorophore 1 10 in the detection antibody can then be used to indicate that the protein has been captured, and was therefore present in the sample tested in the assay.
• a convenient multiplexed analyte capture platform can thus be prepared for any desired application, comprising a plurality of sets (or groups) of magnetic particles, each set of particles carrying a unique code and functionalised with the corresponding capture antibody.
• the plurality of sets of particles corresponding to those target proteins can be mixed together in an assay sample, such as a patient sample on which a diagnosis is to be performed using a multichannel assay.
• an analyte reagent consists of a desired set 120 of functionalised magnetic particles carried in a fluid medium 122.
• the analyte reagent may include a set or group of approximately 100-1000 coded magnetic particles (MCs) functionalised with the capturing antibody for each target protein 106.
• MCs coded magnetic particles
• the analyte reagent is mixed with the sample to be analysed, and is allowed to react with any target proteins present.
• the magnetic particles are then removed from the sample and the fluid medium by magnetic separation 124. This involves attracting the particles together using an external magnetic field 126 (for example so that they gather 128 at the bottom of a container holding the sample) and the sample removed or decanted.
• the particles are then re-suspended in a fluid medium 130 and exposed to the corresponding fluorescently-labelled detection antibodies 108.
• the particles are then driven, or steered, and positioned on a surface 132 for reading.
• the surface may be a glass slide for example.
• each particle has been magnetised out-of-plane, with the magnetisation in a unique direction towards or away from the top surface of the particle, the particles can be steered so that they are all in the same plane as each other, on the surface for reading, and so that they are all oriented in the same way, for example with the top surface of each particle facing away from the reading surface.
• a significant feature of the multichannel analysis enabled by the barcoding of the particles is that the potential number of plex channels is extremely large, up to as many channels as can be coded by the barcodes, which may even be 1000 channels or more. At the same time, the particles in individual channels can be unambiguously identified, achieving little or no crosstalk between the channels.
• conventional bead based bioassays use fluorescence-based channel identification systems which are much less resistant to crosstalk. For example, one prior art system uses ratios of fluorophores for barcoding beads, and a fluorophore-labelled antibody as the positive signalling for analyte detection. This creates challenges in reliability of channel identification and severely limits plex numbers.

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