Magnetic particles and methods

Abstract

To provide a bioassay with magnetic particles, and a production method thereof.SOLUTION: A magnetic particle 30, 70 has a layered structure 6, 56 between a top surface of the particle and an opposed bottom surface of the particle. Layers of the structure include one or more nonmagnetic layers and one or more magnetized layers. The ratio of a lateral dimension of the one or more magnetized layers to the aggregate thickness of the one or more magnetized layer is greater than 500. Such magnetic particles 30, 70 can be functionalized and marked with readable codes 16, 66 corresponding to the functionalization, for use for performing assays such as bioassays.SELECTED DRAWING: Figure 3a

Description

[Technical field]

[0001] The present invention relates to magnetic particles and methods of making and using magnetic particles. The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 77 9822). [Background technology]

[0002] The technique of using an applied magnetic field to apply mechanical forces to individual magnetic particles has been used in a variety of applications. It is used for iotechnology purposes.

[0003] One of the most important commercial applications of magnetic nanoparticles and microparticles today is the separation of biomolecules. and a bioassay for identifying superparamagnetic iron oxide nanoparticles (SPIONs). has traditionally been used in this commercial range of applications because it provides the property of moving toward an external magnetic field source. This allows the particles to be directed to the desired location for reading the assay information. These particles can be manipulated by applying an external magnetic field. Nanoparticles (diameter 5-20 nm) made by chemical methods. As the particles grow larger, they become ferromagnetic, and the stray magnetic field of one particle can be undesirably attracted to other particles. The effect of magnetic fields on the magnetic field can lead to magnetic aggregation of particles, which can prevent their use in bioassays. The size of these particles is limited by the fact that

[0004] Engineers developing magnetic particles need to optimize the magnetic properties of the particles for each application. Magnetic nanoparticles are now being developed for all of these different applications, including bioassay applications. It has been recognized that a highly desirable property of the electrons is a zero net magnetization remanence state to avoid particle agglomeration. It is being considered.

[0005] A zero net magnetization remanence state is one in which a magnetic particle has no net magnetic current in the absence of a magnetic field. In use, magnetic particles are typically immersed in a liquid. or suspended in a fluid medium and free to move within that medium. Has a nonzero residual moment For particles that are in contact with each other, the stray magnetic fields of the particles can interact and cause aggregation or aggregation of the particles. The purpose of using magnetic particles in biotechnological applications is to detect the magnetic fields by applying an external magnetic field. can manipulate or induce the movement of particles suspended in a liquid or fluid medium. This is undesirable because it would result in the magnetic particles clumping together. do not have.

[0006] Additionally, it is well known in the art that small magnetic fields from the environment can induce magnetic moments in magnetic particles. In order to prevent the induction of aggregation, the net magnetization remanence state is It is important to ensure that the zero particles also have low magnetic susceptibility in small magnetic fields. It should be understood that:

[0007] Additionally, when sensitive particles are used, an applied electric field can be applied to induce the particles in the desired manner. After being guided or moved, particles that aggregate during the application of the electric field will agglomerate again when the applied magnetic field is removed. This should also be avoided for magnetic particles in biotechnology applications. This is understood by those skilled in the art.

[0008] Therefore, previous research in the technology of creating non-aggregated magnetic nanoparticles has yielded zero positive results. We have focused entirely on systems with a remanent magnetization state, preferably a low magnetic susceptibility. These include superparamagnetic nanoparticles, magnetic vortex micro- and nanoparticles, and antiferromagnetic coupling. Micro- and nano-particles that utilize magnetization to create opposite magnetization configurations between adjacent magnetic layers within a particle This includes various systems such as

[0009] An important biotechnology application is multiplexed immunoassay of biological samples. Accurate quantification of proteins in biological samples is essential for research and Multiplex immunoassays are of great importance for both clinical and diagnostic applications. This method quantifies multiple different proteins simultaneously in a given sample. Analyzing protein fingerprints could accelerate research and improve diagnostics. In response to this market acceptance, Luminex (RTM), Firefly (RTM Multiplex assay systems such as the Fireplex (RTM) and the ELISA kit have been developed. In these systems, each particle is coated with a capture antibody appropriate for one specific analyte. Then, combine multiple sets of analyte-specific particles to create a single particle set. In combination, multiple targets can be detected simultaneously using fluorescently labeled detection antibodies. The Luminex (RTM) system can be used to measure and quantify polystyrene or It is based on paramagnetic microspheres, or beads, and separates one set of beads from another. They are internally stained with red and infrared fluorophores of different intensities to distinguish them from each other. The Firefly (RTM) and Fireplex (RTM) systems also use fluorophores. Using a filter, it is possible to distinguish one set of particles from another. In the case of , particles are rod-like encoded by applying different fluorophores to each end. The measurement of the fluorophores allows one rod to be distinguished from another. The purpose is to:

[0010] In practice, however, these systems have limited the ability to multiplex assay results. Limited multiplexing due to limited ability to reliably discriminate between channels However, they suffer from a limited number of different proteins that can be synthesized. Summary of the Invention

[0011] The present invention relates to magnetic particles, a plurality of magnetic particles for carrying out an assay, and the magnetic particles referred to herein. As defined in the accompanying independent claim, the present invention provides a method for carrying out an assay using magnetic particles. Preferred or advantageous features of the invention are set out in the dependent claims.

[0012] Thus, in a first aspect, the present invention provides a layer between a top surface of a particle and an opposite bottom surface of the particle. The magnetic particles may be provided with a magnetic structure, the layer comprising one or more magnetized layers. The ratio of the lateral dimension to the thickness or total thickness or effective thickness of the magnetic layer or layers is , greater than 500. In other words, the aspect ratio of the cross section of the magnetization layer is greater than 500. In preferred embodiments, the ratio may be higher, for example, greater than 800. or greater than 1000 or 1500 or 2000.

[0013] The particles may further comprise a non-magnetic layer, which may advantageously provide mechanical support to the magnetic layer, The physical properties of the particles, such as the mechanical properties and density, can be determined.

[0014] The particles may comprise one magnetizable layer, or may comprise two or more such layers. When the magnetic layers are provided, the layers may be adjacent to each other, may be in contact with each other, or may have non-magnetic material between them. The total thickness or sum of the remanent magnetic layers in a grain having two or more magnetized layers may be The total thickness may be the sum of the thicknesses of the magnetic layers, not including the non-magnetic layers between them. In some embodiments, the layered structure may include many magnetic layers and / or many layers of non-magnetic material. The layer may include

[0015] The magnetization or magnetic layer may be, for example, a ferromagnetic material, an element or alloy, or a superparamagnetic nanoparticle. The magnetic material may comprise any suitable magnetic material or materials, such as a composite of

[0016] The particles are preferably substantially flat in shape and have magnetic and and / or one or more substantially flat layers of non-magnetic material. Substantially the same shape and size, each having the same lateral shape and size as the particle itself. However, the shape and structure of the particles may vary, as will be further explained below. may differ from the above.

[0017] The particles may have zero or non-zero remanence. However, the inventors believe that the particles Even if they have a non-zero remanence, the shape and structure of the particles embodying the present invention may be such that the surface of the particle is The surface exhibits an unexpectedly low stray magnetic field, thereby allowing the It has been discovered that a number of particles may advantageously not aggregate or clump together. Surprisingly, the inventors have found that this occurs regardless of whether the particles are completely magnetoremanent. We found that this was the case.

[0018] In a preferred embodiment of the present invention, when an external magnetic field is applied, the particles The magnetic moment is sufficient to exert a desired force on the particle, the desired force depending on the application of the particle. This may depend on the particle size (e.g., bioassays where particles are suspended in a fluid medium). The applied external magnetic field is typically less than 2T or 1T or 0.5T, typically less than 0.05 It may be greater than T or 0.1T or 0.25T.

[0019] For example, in biological or chemical assays, external magnetic fields are used to manipulate particles within a fluid medium. It may be desirable to use a magnetic field. The magnetic moment may be due to the magnetization of the particle itself. It can also be induced in the particle by an external magnetic field, provided that the particle contains sufficient magnetic material. , it is important to generate the necessary force.

[0020] The magnetic moment generated by an external magnetic field applied to a particle is the It depends on the total volume V multiplied by the magnetization Ms of the material. The V.Ms value of the particles is preferably 10 -18 J / T or 5×10 -18 J / T or 10 -17 It is greater than a given value, such as J / T.

[0021] The inventors have demonstrated that the physical distribution of magnetic material within a particle can determine the stray magnetic field near the particle. Thus, it has been discovered that it is possible to determine the tendency of particles to interact with one another and / or to aggregate. The present inventors have found that one or more layers (preferably flat) having a cross section with a high aspect ratio AR. By distributing the magnetic material in the form of a magnetic layer (rows), it is possible to generate an advantageously low stray magnetic field. This preferred particle shape has the advantage of having a low stray magnetic field and low tendency to agglomerate. The particles may be provided with little or no

[0022] In order to evaluate this particle shape more quantitatively, the inventors have determined that the magnetic Parameter AR / Ms of the magnetic layer or layers 2 (The dimensionless ratios AR and A / m( 1000A / m is 1emu / cm 3 ) in the magnetic layer or in multiple magnetic layers The magnetization of a magnetic material (Ms) is 8x10 -10 m -2 / A -2 , or 1. 2x10 -9 m -2 / A -2 or 8x10 -9 m -2 / A -2 Greater than.

[0023] Alternatively, or in addition, the inventors recommend that the parameter AR / Ms is preferably 1x10 -3 m -1 / A -1 or 3x10 -3 m -1 / A -1 or 5x10 -3 m -1 / A - 1 It was decided to be larger than

[0024] These limits are approximately 2500 A / m( Depending on the particle's application and environment, it may be possible to tolerate this level of stray magnetic field. The field may advantageously prevent agglomeration.

[0025] When evaluating the AR value of a grain structure, the AR is calculated by dividing the cross-sectional lateral dimension of the structure by the thickness of the structure. For a layer of magnetic material, the lateral dimension may be the smallest lateral dimension of the layer. However, when the layer shape is more complex, it may be preferable to consider the average lateral dimension of the layer. If the thickness of the layer is constant, the thickness can be used to calculate the AR. If the thickness of the layer is different, In this case, the average thickness may be used.

[0026] For example, if the magnetic material extends across the lateral dimension of the grain, the grain may be magnetized, especially if the grain thickness is too large. If the thickness of the magnetization layer or layers is sufficiently similar, the thickness of the magnetization layer or layers 10 times or 5 times, for example, using the equivalent lateral and thickness dimensions of the particle itself. It may be appropriate to use this approach to estimate AR / Ms. In this study, the value of Ms is calculated using the AR value of the particle as well as the M of the magnetized material within the particle. By calculating the thinning Ms value, s multiplied by the volume ratio of non-magnetized and magnetized material in the particle It may be changed.

[0027] The particles may comprise a plurality of magnetic particles, for example in the form of a stack of magnetized layers separated by layers of non-magnetic material. In such a case, the magnetization layer may be 5 times the thickness of the thinnest layer or the average layer thickness. , 10x, or even 20x, the AR is The total thickness of the magnetic layers or the distance between the outermost magnetic layers in a stack (including the thickness of the intervening non-magnetic layers) (including

[0028] If a particle comprises multiple layers spanning a sufficient fraction of the particle's thickness, the AR can be calculated by multiplying the thickness of the particle by It can be evaluated using.

[0029] If a particle comprises multiple layers, an alternative approach to estimate the thickness for calculating the AR is The approach can be to calculate the thinning thickness of the magnetized material. For example, the magnetization If two or more parallel layers of a material are separated by a layer of non-magnetic material with a total thickness of T nm, then The thinning thickness of the thinned material is Tm / (Tm+Tnm).

[0030] The measurement reveals that the AR and Ms (AR / Ms 2 or for the calculation of AR / Ms) It may also be possible to evaluate the magnetic field of a given number of particles of unknown structure but known dimensions. When measuring moment (e.g., when using a vibrating sample magnetometer), the total particle volume and The total moment per particle can be used to determine the effective Ms. The dimensions and thickness can be determined, for example, by microscopy and / or electron microscopy to assess the AR. It can be measured directly using inspection techniques.

[0031] Particles embodying the invention are preferably planar in shape and have a length and width Both are greater than their thickness. Advantageously, the length and width of the particles, or perpendicular to each other, The two lateral dimensions of the particle measured directly are similar to each other or are approximately 10 times smaller than each other. %, 30%, 50%, or 70% difference. The particles are usually round, elliptical, or or a polygonal disk, or an approximately flat rectangular prism having a square or rectangular perimeter. It could be.

[0032] The magnetic material within the grain is distributed across the lateral dimensions of the grain in the form of a layer or layers within the grain. Preferably, the magnetic layer or layers have an aspect ratio of: The minimum lateral dimension of a layer or layers, which may be equal to or smaller than the lateral dimension of a particle. The magnetic layer may be evaluated by reference to its longitudinal dimension or its average lateral dimension. The aspect ratio, AR, is the ratio of the minimum or average lateral dimension of a magnetic layer to its thickness (or the thickness change). If multiple magnetized layers are present, the aspect ratio AR is the lateral dimension, or the average lateral dimension if different layers have different lateral dimensions. The lateral dimension can be evaluated as the thickness of the layer divided by the total thickness of the layer.

[0033] In a preferred embodiment, the top and bottom surfaces of the particles are 5 nm, or 10 nm or From 50nm or 100nm to 100μm or 50μm or 5μm or 1μm The particles may be separated by a particle thickness of between 1 μm and 500 nm. m, preferably greater than 5 μm or 10 μm, with a maximum lateral dimension of 500 μm or less The thickness of the particle may be less than 200 μm, 100 μm, or 50 μm. the ratio of the smallest lateral dimensions of the particles is greater than 10 or 20 or 50; and / or It may be less than 2000, 1000 or 500. Therefore, such a preferred In embodiments, the particles can have fairly flat, high aspect ratio shapes, while others can have large, Embodiments include the use of lower aspect ratio particle shapes, and even spherical or cubic shaped particles. Such lower aspect ratio particles are contemplated as discussed above and herein. In one embodiment, the magnetic layer may have one or more magnetic layers with a higher aspect ratio so that the magnetic layer can be made to have a larger aspect ratio.

[0034] In embodiments with multiple magnetized layers, the layers are preferably substantially parallel to one another. In embodiments with multiple magnetizable layers, the layers are preferably similar to each other. They have a shape and / or area and can conveniently overlap one another, and in some cases completely overlap one another. may overlap.

[0035] The opposing top and bottom surfaces of the particles are advantageously flat, but may be agglomerated if desired. without affecting the desired properties of the particles with a stray magnetic field small enough to avoid , one or both sides may be curved and may not be flat. Thus, the particle itself It may be flat or curved, but in either case the opposing top and bottom surfaces are Readable information such as a readable code in the form of a digital code or 2D code Apply the function to the top and / or bottom surfaces to filter individual particles or groups of particles (particle groupings). If the particles in the loop are similarly marked, they are identified by reading the information Such information may advantageously have a sufficiently large area so that it is possible to may be applied to the top and / or bottom surface, or may be applied below the top and / or bottom surface. For example, the surface layer or layers may be coated through the surface layer or layers. The cover or information is sufficiently transparent so that the cover or information can be read. The bottom surface is advantageously biofunctionalized or bioresorbable for the biotechnological or chemical applications described herein. These particles may form a suitable substrate for applying other functionality, such as nanoparticles or chemical functionality.

[0036] Thus, in a preferred embodiment, the shape of the particles is a high aspect ratio rectangular or rectangular solid. It may be in the form of a thin (thin) laterally extended shape, such as a disk. The area ratio refers to the ratio of the smallest or average lateral dimension to the thickness. In this case, the magnetic particles can be described as cylindrical, with the thickness of the particle being along the axis of the cylinder and the circumference of the cylinder being along the axis of the The edge shape is preferably free of reentrant angles, typically with convex or straight edges. The preferred perimeter shapes are rectangular, square, or circular. It is.

[0037] As mentioned above, the particles are preferably of high aspect ratio or cylindrical shape, with top and bottom Although the surface is flat, embodiments of the present invention achieve the objective of providing non-agglomerated magnetic particles. However, curved or non-flat particles, or particles having curved or non-flat upper and lower surfaces, It is believed that the particles may include

[0038] Preferably, the difference between the smallest and largest lateral dimensions of the magnetic particles is less than 90% or less than 70%. In a preferred embodiment, the particles have a minimum lateral dimension greater than 5 μm, preferably Preferably the particles are larger than 10 μm and / or the maximum lateral dimension is less than 500 μm. These dimensions are less than 200 μm, 100 μm or 75 μm. The selection may be made by one skilled in the art depending on the requirements, such as the intended use and the desired mechanical strength of the particles. .

[0039] The layer structure of the magnetic particles advantageously comprises a magnetizable layer and a non-magnetic layer. The non-magnetic layer can provide sufficient strength and provide a suitable substrate for the magnetizable layer. Al, Ta, Pt, Pd, Ru, Au, Cu, W, MgO, Cr, Ti, Si, Ir, S iO2, SiO, Sn, Ag, polymers, plastics, alloys of these materials, and It may advantageously comprise a material selected from composites or mixtures including these materials.

[0040] The particles may comprise two or more layers of non-magnetic materials which may also be selected from this group.

[0041] The magnetic or ferromagnetic layer may be formed from any suitable material, and in a preferred embodiment, for example For example, Fe, Co, Ni, CoFe, CoFeB, FePt, CoNi, and NiFe The material may comprise a material selected from metals or metal alloys such as:

[0042] The magnetization layer may be, for example, a magnetic multilayer of alternating layers of magnetic material and precious metals (e.g. Pt / CoFeB). The pair may comprise a tack, the pair being known to provide perpendicular magnetic anisotropy.

[0043] The magnetization layer is preferably an out-of-plane magnetization layer, but can be a different magnetization layer, such as an in-plane magnetization layer. .

[0044] To achieve a rapid response to an external magnetic field, the magnetic particles must have a high saturation magnetic field. A magnetic material is selected to achieve this.

[0045] The layer structure of the magnetic particles may comprise multiple layers of non-magnetic material and / or multiple layers of magnetizable material. In a preferred embodiment, the particles are located between two layers of non-magnetic material. The magnetic layer may include a magnetic layer.

[0046] The particles are arranged in pairs such that the remanence is zero when no magnetic field is applied. Such particles may comprise two or more magnetizable layers, each of which is connected to a magnetic field. Thus, for example, the particles may be attracted to the magnetic field through a fluid medium. An external magnetic field may be applied to move or manipulate the particles. It may not matter whether the particles are highly or less sensitive. The shape is such that the stray magnetic field around the particles causes the particles to aggregate even when a magnetic moment is induced. This is because the probability of this happening is too low to be favorable.

[0047] Advantageously, in such particles, the magnetizable layer extends from the upper surface of the particle through 25% of the thickness of the particle. and from the base of the particle may be spaced more than 25% of the thickness of the particle. The structure advantageously further reduces stray magnetic fields at the opposing top and bottom surfaces of the particle. obtain.

[0048] Preferably, the magnetic layer is greater than 0.1 nm, or 0.4, 1.0, or 1.5 nm. Preferably, the thickness of the magnetic layer is less than 2 times the total thickness of the grain. It is preferred that the mechanical strength of the particles is low, preferably low, such as less than 5%, more preferably less than 15% or even 10%. If this is sufficient for the application, the particles may comprise only a magnetizable layer.

[0049] The magnetizing layer can be, for example, a thin film multilayer.

[0050] at or within a short distance of the top or bottom surface of the particle, averaged over the entire side The net magnetic field (stray magnetic field) is preferably less than 2500 A / m (300e), particularly preferably It can be less than 800 A / m (10 Oe) or 400 A / m (5 Oe). At the surface or at a short distance, such as 10 nm, 50 nm or 100 nm from the surface, e.g. The inventors' experiments have demonstrated that these external or stray magnetic fields can be used to measure the magnetic field. It is shown that the field is small enough to avoid aggregation of the magnetic particles.

[0051] Magnetic particles embodying the present invention are conveniently manufactured or fabricated by lithographic processes. It can be done.

[0052] The second aspect of the invention may advantageously provide magnetic particles having the above dimensions, preferably The top surface of the particle and the bottom surface of the opposite side of the particle are separated by a particle thickness of 5 nm to 200 μm. the particles have a minimum lateral dimension greater than 1 μm and a minimum lateral dimension relative to the thickness The ratio of the number of residual particles to the number of residual particles is greater than 10, where the particle has a layered structure throughout its thickness, one or more residual particles It comprises a magnetic or magnetized layer and one or more layers of non-magnetic material. Such particles can be conveniently manufactured by lithographic processes and can be used in the present invention as described herein. It may comprise one or more features of the first aspect of the invention.

[0053] In a further embodiment of the invention, the top or bottom surface of the particle is provided with a readable code, e.g. It may carry readable information, for example a bar code or a 2D code. This allows the information to be read, for example, using a camera and suitable software. By doing so, it may be possible to identify particles remotely.

[0054] In a preferred embodiment, the magnetic properties of the particles allow for the particles to be magnetically coupled to the fluid medium by application of an appropriate external magnetic field. A position for manipulating, moving or driving particles through a For example, a large object carrying code or information can be moved to a specific location. Particles with high aspect ratio shapes, with large top or bottom surfaces, tend to form nanoparticles that are in contact with a substrate or other supporting surface. Thus, the code or information can be conveniently guided to be read.

[0055] In a further embodiment of the invention, the top and / or bottom surfaces of the particles are functionalized, e.g. The particles can be physically or chemically functionalized, which allows for the application of readable information to the particles. For example, the top or bottom surface of the particle may be readable. The substrate may carry a code that can be functionalized on the same or opposite surface. In this embodiment, a plurality of particles, each of which carries readable information corresponding to the functionalization of that particle, are may be provided.

[0056] Such particles can be used to provide the particles to a liquid or fluid assay sample and to detect the functionality of the particles. interacting with the assay sample, e.g., with a biomolecule or other component of the assay sample By applying a magnetic field, it may be possible to carry out assays such as bioassays. The particle is manipulated into a reading position, the readable code is read, and the particle's function and By measuring the interaction with the sample, an assay result can be obtained.

[0057] Multichannel assays allow each particle to have a readout that corresponds to a different function of that particle. This can be done by providing a plurality of particles carrying the information. loops, with each group of particles carrying the same readable information and retained in the same way. The plurality of particles may be contacted with a liquid or fluid assay sample, and the functionality of the particles may be determined by the assay sample. A magnetic field is applied to manipulate the particle into a read position. The readable information of two or more particles is read, and the corresponding function and assay sequence of each particle are obtained. The assay result is obtained by measuring the interaction of each with the sample.

[0058] Identification of particles using such readable information allows particles to be accurately distinguished from one another. For example, barcode or The 2D code allows the identification of different particles to be easily integrated into existing multiplex assay systems. This provides a much more robust process than the conventional fusion platform, and allows for cross-linking between plex channels. Loss of control is minimized. Moreover, the use of readable information in this way This allows for many more multiplex channels than are currently possible. For example, a barcode or 2D code can be 1000 plex, or 10, 000 plex, or more as needed.

[0059] Thus, in a preferred embodiment, the present invention provides a method for the preparation of ... compound having a molecular weight of 100 or more for use in biotechnology applications. For this purpose, advantageously ferromagnetic microdisks (microparticles, nanoparticles, microcarriers, etc.) ) and perpendicular (or out-of-plane) magnetized grains. For example, these particles can be fabricated by photolithography and physical synthesis of magnetized thin-film multilayers. Ferromagnetic microparticles or microdisks (each lateral dimension 1 to 500 μm, or 1 to 200 μm in either or both orthogonal lateral dimensions m, or preferably 5 to 100 μm, and 10 nm to 200 μm, or preferably For example, the particles can be round or square. , the diameter or length of one side may be 40 μm, and the thickness may be 100 nm. The resulting high planar aspect ratio may be 100 μm on a side and 1 μm thick. Ratios, ultra-thin disks, or microdisks (with functions such as biofunctional antibodies for diagnostic tools) The magnetic carrier (MC), which can be called a magnetic carrier because of its ability to hold high magnetic field, The MC is ferromagnetic with a junction. The MC can be defined lithographically. The MC is a ferromagnetic material that is formed on each magnetic layer. aspect ratio (typically 1 nm or 5 nm for the total magnetic layer thickness and tens of lateral dimensions μm) and the magnetization direction perpendicular to the plane of the MC is such that the stray magnetic field from each particle can be neglected. Therefore, they do not aggregate when suspended in a fluid.

[0060] In a preferred embodiment, the MC has a magnetization direction parallel to the surface normal of the microdisk, as well as These properties can be characterized by high magnetic anisotropy and high coercivity reversal. High degree of control over the magnetic response under influence and therefore over the mechanical behavior in the fluid This may allow for control.

[0061] The physical vapor deposition process preferably used to manufacture the particles or MCs is This allows for sub-nm control in the deposition of magnetic thin films, thus enabling the engineering of the magnetic properties of MCs. This provides extremely high precision in geneering. In addition, barcode (or other readable) Information of interest can be added lithographically to the surface of the MC for optimal functionalization with molecules of interest. The surface material may be selected accordingly. [Brief description of the drawings]

[0062] Specific embodiments and best mode of the present invention Specific embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] FIG. 2 shows steps in two processes, Process A and Process B, for the manufacture of magnetic particles according to the first and second embodiments of the present invention. [Diagram 2]FIG. 1 shows polar magneto-optical Kerr effect (MOKE) measurements of the magnetic response of the magnetic thin film 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) / CoFeB(0.6) / Pt(5.0) used in the particles of the embodiments. [Figure 3a] FIG. 1 illustrates how a readable code according to an embodiment and the magnetic states within a particle are linked by an applied magnetic field to ensure that the readable code can always be aligned with an external detector such as a camera or barcode reader, and shows an image of the readable code of a particle imaged by a detector. [Figure 3b] FIG. 1 illustrates how a readable code according to an embodiment and the magnetic states within a particle are linked by an applied magnetic field to ensure that the readable code can always be aligned with an external detector such as a camera or barcode reader, and shows an image of the readable code of a particle imaged by a detector. [Figure 4] FIG. 13 illustrates magnetic stray field strength as a function of distance from a surface of particles produced in accordance with an embodiment. [Figure 5a] FIG. 1 shows functionalized particles according to further embodiments of the invention suitable for bioassays and illustrates the use of particles according to further embodiments of the invention for performing streamlined multiplex assays. [Figure 5b] FIG. 1 shows functionalized particles according to further embodiments of the invention suitable for bioassays and illustrates the use of particles according to further embodiments of the invention for performing streamlined multiplex assays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] A particular embodiment of the present invention is a high-performance ferroelectric thin-film ... This includes the production of magnetic moment microparticles. The high aspect ratio of the shape of these particles allows This results in a very low stray magnetic field from each particle, and magnetic nanoparticles do not exhibit interparticle interactions. (Therefore, they do not aggregate.) When an external magnetic field is applied, the particles transition to magnetic saturation and are forced Each barcode is created using a simple and robust lithography technique. It can be added to the particles using a graphic process and read optically. Thus, magnetic particles can be used to perform robust multiplex assays, such as cytokine assays. The results demonstrated highlight their potential in assay applications.

[0064] In this embodiment, the lithographically produced magnetic particles have a high magnetic moment, interparticle Advantages: no interactions, large surface area for functionalization, and robust particle-specific barcodes These particles have the ability to both functionalize and carry readable information. Considering the large surface area of ​​the particles, they can be called magnetic carriers (MCs). Advantageously, it may provide a larger area for functionalization than particles.

[0065] Lithographically defined magnetic nanoparticles are known in the prior art, for example from: T. Vemulkar, R. Mansell, DCMC Pet it, RP Cowburn, and MS Lesniak, “High hly tunable perpendicularly magnetized s synthetic antiferromagnets for biotechnol ogy applications,” Appl. Phys. Lett., 20 15, H. Joisten et al., “Self-polarization pheno menon and control of dispersion of synthesis etic antiferromagnetic nanoparticles for biological applications,” Appl. Phys. L ett., vol. 97, no. 25, p. 253112, 2010, S Leulmi et al., “Comparison of dispersion and actuation properties of vortex and synt hetic antiferromagnetic particles for bi otechnological applications,” Appl. Phys. Lett., vol. 103, no. 13, p. 132412, 20 13 However, these lithographically defined particles and general magnetic nanoparticles are not comparable. In contrast, the MC used here maintains a zero net remanent magnetization state to prevent particle aggregation. As used herein, MC refers to a net zero residual quantity, if necessary. May have residual magnetism (and susceptibility to the generation of magnetic moments in external magnetic fields) However, despite the conventional expectations of those skilled in the art, they have zero net remanence to avoid agglomeration. The stray field of a particle determines whether its remanence in the absence of an external magnetic field is zero or not. Regardless of whether the magnetization is performed by a particle, the shape of the magnetized material within the particle and / or the shape of the particle must be avoided to avoid agglomeration. low enough to

[0066] The MC in this embodiment is a rectangular parallelepiped with a very high aspect ratio, with the length and The thickness of the layer is approximately 150 nanometers and the width is 40 microns.

[0067] Two lithographic methods according to two embodiments of the invention for producing magnetic particles or MCs. The process (A and B) is shown in FIG.

[0068] Process A is shown in Fig. 1.A1 to Fig. 1.A11. In Fig. 1.A1, 5 A 0 nm sacrificial layer 2 is grown by magnetron sputtering on a Si substrate 4. Next, the base 6 of the particle thin film stack is also deposited by magnetron sputtering. It grows on a sacrificial layer.

[0069] The base is Au(100.0) / Ta(2) / Pt(4) / CoFeB(0.6). / Pt(1.2) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / P It is composed of 11 layers (thickness: nm) of t(1.2) / CoFeB(0.6) / Pt(5.0). It is being done.

[0070] In FIG. 1.A2, photoresist 8 is spin-coated onto the MC base 6 The photoresist is then coated with a particle barcode (or readable code) as shown in Figure 1.A3. A photomask 10 defining a pattern (a region) is used to expose the pattern during lithographic patterning. This standard photolithography process is repeated on the photoresist shown in Figure 1.A4. 1, creating a number of holes 12 where a barcode contrast material 14, such as 15 nm of Ta, is placed. , deposited on the particle base using magnetron sputtering, as shown in Figure 1A5. The shape and pattern of the holes defines the bar code 16.

[0071] Next, the photoresist is removed with a solvent such as acetone, and the photoresist is removed by photolithography, as shown in Figure 1.A6. A new layer of resist 18 is spin-coated onto the particle base 6 and barcode 16. This is done by a second lithography step using a mask 20 to define the shape of the particles. In this step, shown in Figure 1.A7, the particle shape is imaged. A plurality of holes 22 defining the bar code are aligned so that the bar code is aligned with the center of the holes. do.

[0072] In Figure 1.A9, particle (MC) cap 24 and ion beam milling hard mask 26 is a magnetron consisting of 30-40 nm Au and 200 nm Al, The thickness of the gold is added by sputtering. The thickness of the gold is determined by the presence of particles (top and bottom) for biocompatibility. Both surfaces (top and bottom) were completely coated with Au and were chosen to provide a surface for biofunctionalization. However, the thickness of the Au is thin enough that the barcode can be read through the Au layer. It is possible.

[0073] In Figure 1.A9, the photoresist 18 is removed with a solvent such as acetone, leaving the entire sample is subjected to ion beam milling, a standard subtractive patterning process. All thin films not protected by the ion beam milling hard mask are milled away. Thus, milling removes particles that are not within the defined grain shape from the thin film stack. Remove all the thin films that form the base. When the sacrificial layer is reached, the milling process stops. The remaining Al hard mask 26 is removed by 3 to 5% tetramethylammonium hydroxide solution or can be removed by dissolving in an equivalent Al solution etchant for 10 to 30 minutes. do.

[0074] In this way, photolithographic patterning determines the planar shape of the particles, and physical vapor deposition determines the shape of the particles. The deposition process determines the thickness and composition of the particles.

[0075] At this stage, the barcode-bearing particle 30, MC, is fully defined and rests on the sacrificial layer. Next, a magnetic field 32 larger than the coercive field for the magnetic thin film of the particles is applied, as shown in FIG. As shown in Fig. 1, all particles are in the "up" state, perpendicular to the top and bottom surfaces of the particle. , ensuring that the particles are magnetized out of plane. Alternatively, the particles are all magnetized in the "down" state. This links the magnetization of the grains to the physical structure of the grains in the perpendicular direction, as shown in Figure 3. This allows for the alignment of barcodes in downstream steps such as redeposition or analysis in solution. do.

[0076] Finally, the sacrificial layer of Al underneath the particles was dissolved in a suitable solvent to form a , lifting the particles 30 from the substrate and releasing them into solution in the fluid medium.

[0077] Process B is shown in Figures 1.B1 to 1.B11. In Figure 1.B1, the photo A resist layer 50 is spin-coated on a Si substrate 4. Next, in FIG. 52 is used to expose the photoresist 54 to light. A pillar is created on which a series of layers of material 56 are magnetron sputtered in FIG. 1.B3. The layers are deposited using a metal sputtering process to form a thin film structure base 58 of layered magnetic particles. The shape of the band or pillar defines the shape of the particle. The structures after deposition of the first layer are shown below. a(2) / Pt(4) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / It is composed of a thin layer of Pt(5.0) (thickness is nm).

[0078] A lithographically defined barcode is then added to the particle. A second layer of photoresist 60 is applied and patterned with the desired bar code for each particle. A patterned photomask 62 is used to expose as shown in FIG. 1.B5. In FIG. 1.B6-7, the photoresist is developed and flood exposed 64 to a downstream developer. The bottom of each photoresist island or pillar 54 can be removed with a liquid. The layer is shielded from this exposure step by the presence of particles on top of the islands. .

[0079] A barcode contrast material such as 15 nm of Ta is formed on top of the particle in Figure 1.B8. The top layer of photoresist is then completely removed using a developer in Figure 1.B9. The resist is then removed and a particle cap 68 of 30-40 nm Au is deposited. The bottom layer remains intact. The gold thickness is reduced by MC (top and bottom surfaces) with Au for biocompatibility. The substrate is selected to completely coat the surface (both surfaces) and provide a surface for biofunctionalization. However, the thickness of the Au is thin enough that the barcode can be read through the Au layer. It is.

[0080] In this way, photolithographic patterning determines the planar shape of the particles, and physical vapor deposition determines the shape of the particles. The deposition process determines the thickness and composition of the particles.

[0081] At this stage, the barcoded particles, MC,70, are fully defined and in the photoresist. Next, a magnetic field 72 is applied that is greater than the coercive field for the magnetic thin film of the grains. In addition, as shown in Fig. 1.B10, all grains are in the out-of-plane “up” state, and the grains Ensure that the particles are magnetized out of plane, perpendicular to the top and bottom surfaces. Alternatively, all particles are magnetized “down This links the magnetization of the grains to the physical structure of the grains in the perpendicular direction. The barcodes are then aligned in downstream steps such as redeposition and analysis in solution, as shown in Figure 3. It will be possible to do this.

[0082] Therefore, the thin film structure of MC described in this embodiment in processes A and B is 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) / C The base is defined as FeB(0.6) / Pt(5.0) (thickness in nm). 1 A 5 nm Ta barcode sits on top of this layer, which is then capped with 30-40 nm of Au. Because the Au on the surface is thinner, it is possible to image the barcode through the Au, Thus, in the embodiments described herein, the barcode is only visible from the top surface of the particle. Therefore, at this point it is not possible to relate the magnetization of the particles to their physical structure, since the particles are in solution. This is necessary to allow control and orientation of the child's barcode surface.

[0083] Finally, the photoresist 74 under the particles is dissolved in a suitable solvent, as shown in Figure 1.B11. This lifts the particles 70 off the substrate and releases them into solution in the fluid medium.

[0084] FIG. 2 shows the magnetic thin film 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) / CoFeB(0.6) / Pt(5.0) The magneto-optical Kerr effect (MOKE) measurement of the magnetic response. The magnetization of the thin film is clearly out of plane. The magnetic field is off and a sudden, forced magnetic transition to saturation occurs. H C is the coercive field, or the thin film The magnetic field required to switch the magnetic field to the saturated magnetic state is shown.

[0085] FIG. 3a shows how the magnetic state of the barcode (or other 2D code) and the particles 30, 70 are to ensure that the code is always aligned to the external detector. First, as mentioned above, before the particles are lifted into the solution, H C Magnetic field exceeding The magnetization of the particles is set to the "up" state using the The particles retain this magnetization state unless exposed to a magnetic field. C Less than When this happens, the particle simply rotates to align its magnetic moment M with the externally applied magnetic field. A field strength of 10 to 1000 Oe and a frequency of 0 to 50 Hz are usually sufficient to induce particle motion. The barcodes are printed on the top surface of each particle and are 30-40 nm in size. The magnetic fields are coated with Au, through which they can be optically imaged. Aligning the cleavage member M with an external magnetic field aligns the particle barcode in the direction of the applied magnetic field (H). Thus, the particles respond to the applied magnetic field by uniquely aligning the doped surfaces, as shown in Figure 3. The field can be used to guide the light onto a substrate, such as a planar substrate, and can include barcode readers and fluorescent detectors. The signal may be read by an associated detector, such as a sensor or a camera.

[0086] Figure 3b shows an image of a particle on a planar substrate and a readable code such as a barcode. is doing.

[0087] FIG. 4 shows the strength of the stray magnetic field as a function of distance from the surface of the particle 30, 70 of the embodiment. It can be seen that the stray magnetic field is low due to the high aspect ratio geometry of the particles. This advantageously reduces the tendency of the particles to agglomerate.

[0088] The particles according to one embodiment of the present invention are used to perform a multiplex assay as follows: The steps of the process are shown in Figures 5a and 5b. The bar code 102 (preferably a quick code) is produced, for example, by lithography as described above. Patterned with a QR code or 2D data matrix code A predefined barcode, or other readable code, is attached to the multichannel bioassay. The resulting sequence is assigned to a desired assay target, such as a specific protein identified in the assay. Then, each particle carrying the code corresponding to a particular protein is The ELISA kit is functionalized with capture antibodies 104 specific for each of the proteins. This can be carried out using chemical protocols. The gold surface of the particles is suitable for this functionalization.

[0089] In this and other embodiments, if other functionalization of the particles is required, Materials other than gold may be used for either or both of the top and bottom surfaces. For example, SiO2 may be used. Good too.

[0090] Detection of the analyte is performed by a conventional sandwich immunoassay. The capture antibody binds to the target protein. When the protein 106 is captured, the magnetic particles are attached to a fluorescently labeled detection antibody 108 that is complementary to the capture antibody. As will be appreciated by those skilled in the art, exposure to the detection antibody will bind to and label the protein. The fluorescence of fluorophore 110 indicates that the protein is captured and therefore tested in the assay. It may be used to indicate the presence of a specific compound in a sample.

[0091] Therefore, a set (or group) of magnetic particles is paired with a unique code. and a set of particles, each functionalized with a corresponding capture antibody, for any desired application. A convenient multi-analyte capture platform can be prepared for a desired range of target proteins. To achieve this, multiple sets of particles corresponding to the target proteins are analyzed using a multichannel assay. The antibody may be mixed into an assay sample, such as a patient sample for which a diagnosis is to be made using the antibody.

[0092] In the assay according to the preferred embodiment shown in FIG. 5b, the analyte reagent is 22. In a typical example, the separation The analysis reagent consists of approximately 100-1000 antibodies functionalized with capture antibodies for each target protein. The analyte reagent may comprise a set or group of encoded magnetic particles (MC). The sample is mixed with the target protein and allowed to react with any target proteins present. The particles are removed from the sample and fluid medium by magnetic separation 124. An external magnetic field 126 is used to attract the particles together (e.g., to the bottom of a container holding the sample). This involves removing or decanting the samples so that they collect. The particles are then resuspended in a fluid medium 130 and exposed to a corresponding fluorescently labeled detection antibody 108 . The particles are then driven or manipulated by application of an external magnetic field to the surface 132 for reading. The surface can be, for example, a glass slide. In particular, each particle is The magnets are magnetized in a specific direction toward or away from the top surface of the particle. Therefore, the particles are all in the same plane on the surface for reading, and And, for example, each particle is aligned so that its top surface faces away from the reading surface. The two can be manipulated so that they are all oriented in the same direction.

[0093] In an alternative embodiment, the particles may be magnetized in a plane where the magnetic field is parallel to the top or bottom surface of the particle. The particles can be guided to the surface by an external magnetic field for reading, but the top surface of each particle is It is not possible to align all the particles with their bottom faces pointing away from the surface. The apparent problem can be solved in one of two ways. They can be manufactured with double-readable information so that the information can be read from either side. Alternatively, the particles can be, for example, transparent enough to allow the particle layer to be viewed from both the top and bottom. By making the information readable, readable information can be provided on any surface of the particle. can be manufactured so that it can be read from

[0094] Once the particles are placed on the surface 132 for reading, a suitable camera and control software Two images of the particles can be taken using the scanner. The first image 134 shows the code for each particle. This allows the identification of which particles in the image correspond to each target protein. Each particle carries a capture antibody for each channel, or in other words, the multichannel assay to which it belongs. The second image 136 is a fluorescent image of the particles. If the antibody on the particle captures the corresponding protein, the fluorescence intensity is reduced. If a particle does not fluoresce, it may indicate the concentration of the protein in the sample. The proteins that are not captured are not present in the sample. By overlaying the images, each particle can be assigned a fluorescence intensity value, allowing the researchers to identify particles in the sample. The corresponding analysis software can identify the proteins present in the sample. The proteins and the concentrations of those proteins are shown.

[0095] A key feature of multichannel analysis enabled by particle barcoding is the plexing. The potential number of channels is very large, up to as many as can be coded by a barcode. The number of channels can be as high as 1000 or even more. Particles within a channel can be clearly identified, resulting in little or no crosstalk between channels. In comparison, traditional bead-based bioassays Using a fluorescence-based channel discrimination system that is much less tolerant to crosstalk For example, one prior art system uses a ratio of fluorophores to barcoded beads. and use fluorophore-labeled antibodies as positive signaling for analyte detection. This creates challenges in the reliability of channel identification and greatly limits plex numbers. Limited.

Claims

1. A magnetic particle having non-zero remanence and coercivity reversal, comprising: a layer structure between a top surface of the magnetic grain and an opposite bottom surface of the magnetic grain, the layer structure comprising at least two layers, the layers including one or more magnetized layers, the magnetized layer or each of the magnetized layers being an out-of-plane magnetized layer; a ratio of a lateral dimension of the one or more magnetized layers to a thickness of the magnetized layer or to a total thickness of the plurality of magnetized layers is greater than 500; The magnetic particles are magnetized in a direction non-parallel to the top surface or the bottom surface of the magnetic particle.

2. The magnetic particle of claim 1 , wherein the layer comprises a non-magnetic layer.

3. 3. The magnetic particle according to claim 1 or 2, wherein the ratio of the lateral dimension of the one or more magnetized layers to the thickness of the magnetized layer or to the total thickness of the plurality of magnetized layers is greater than 1000.

4. The magnetization layer or layers comprises a volume V of magnetic material having a magnetization or average magnetization Ms, a cross-section of the magnetization layer or layers has an aspect ratio AR, AR / Ms ２ (Ms measured in A / m) is 8 ＊ 10 －１０ (A / m) －２ A magnetic particle according to claim 1 which is larger.

5. The magnetization layer or layers comprise a volume V of magnetic material having a magnetization or average magnetization Ms, the cross-section of the magnetization layer or layers has an aspect ratio AR, AR / Ms (Ms measured in A / m) is less than or equal to 0.001 (A / m) －１ A magnetic particle according to claim 1 which is larger.

6. A magnetic particle according to any one of claims 1 to 5, wherein the top and bottom surfaces of the magnetic particle are separated by a magnetic particle thickness of between 5 nm and 200 μm.

7. The magnetic particle of claim 6 , wherein the ratio of the smallest lateral dimension of the magnetic particle to the thickness of the magnetic particle is greater than 10.

8. 8. A magnetic particle according to claim 6 or 7, wherein the maximum lateral dimension of the magnetic particle is less than 1000 μm.

9. 9. The magnetic particle of claim 8, wherein a minimum lateral dimension of the magnetic particle is at least 10% of the maximum lateral dimension of the magnetic particle.

10. A magnetic particle according to claim 1 , wherein the lateral periphery of the magnetic particle is shaped with convex or straight sides.

11. A magnetic particle according to claim 1 , wherein the lateral dimension of the or at least one of the magnetizable layers is the same as the lateral dimension of the magnetic particle.

12. The nonmagnetic layer may be made of any of a variety of materials, including nonmagnetic metals, nonmetals, semimetals, and compounds, including Al, Ta, Pt, Pd, Ru, Au, Cu, W, MgO, Cr, Ti, Si, Ir, and SiO. ２ , SiO, Sn, Ag, SiN, Ge, polymers, plastics, alloys of these materials, and composites or mixtures thereof.

13. 13. The magnetic particle of claim 1, wherein the or each of the magnetic layers comprises a material selected from magnetic metals, magnetic alloys, magnetic compounds, and superparamagnetic nanoparticle composites, including Fe, Co, Ni, CoFe, CoFeB, FePt, CoNi, NiFe, Fe2O3.

14. A magnetic particle described in any one of claims 1 to 13, wherein the minimum lateral dimension of the magnetic particle is greater than 10 μm.

15. 15. Magnetic particles according to any one of the preceding claims, wherein the or each of the magnetised layers is disposed between the layer of non-magnetic material and a second layer of non-magnetic material.

16. 16. The magnetic particle of claim 15, wherein the or each of the plurality of magnetic layers is located more than 25% of the thickness of the magnetic grain from the top surface of the magnetic grain and more than 25% of the thickness of the magnetic grain from the bottom surface of the magnetic grain.

17. 17. A magnetic particle according to any one of the preceding claims, wherein the or each of the magnetisation layers has a thickness greater than 0.1 nm.

18. 18. A magnetic particle according to claim 1, wherein the thickness of the magnetic layer or the total thickness of the magnetic layers is less than 25% of the thickness of the magnetic particle.

19. 19. A magnetic particle according to any one of the preceding claims, wherein the or each of the magnetisation layers is a thin film multilayer.

20. 20. A magnetic particle according to any one of claims 1 to 19, wherein the net magnetic field (stray field) measured and averaged across the top or bottom surface of the magnetic particle is less than 2500 A / m.

21. 21. Magnetic particles according to any one of claims 1 to 20, produced by lithography.

22. 22. A magnetic particle according to any one of claims 1 to 21, wherein the magnetic particle carries readable information which is readable at or from one or both of the top or bottom surface of the magnetic particle.

23. The magnetic particle of claim 22, wherein the surface of the magnetic particle is biofunctionalized with a biofunctionality that interacts with an assay sample, and the readable information corresponds to the biofunctionality of the magnetic particle.

24. the top or bottom surface of the magnetic particle is biofunctionalized, and the readable information can be read on or from the same surface that is biofunctionalized; And / or the magnetic particles according to claim 23, wherein the surface of each of the magnetic particles is biofunctionalized, and each of the magnetic particles carries the readable information corresponding to the biofunctionality of the magnetic particle.

25. A method for producing magnetic particles according to any one of claims 1 to 24 by a lithographic process.

26. A method for performing an assay comprising the steps of providing magnetic particles according to claim 23 or 24 to an assay sample, interacting the biofunctionality of the magnetic particles with the assay sample, manipulating the magnetic particles to a reading position by applying a magnetic field, and obtaining an assay result by reading the readable information and the interaction between the biofunctionality of the magnetic particles and the assay sample.

27. A method for performing a multi-channel assay, comprising the steps of: providing a plurality of magnetic particles according to claim 24 or 25 to an assay sample; interacting the biofunctionality of the magnetic particles with the assay sample; manipulating the magnetic particles to a reading position by applying a magnetic field; and obtaining an assay result by reading the readable information of two or more of the magnetic particles and the interaction of the corresponding biofunctionality of the magnetic particles with the assay sample.

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