Carrier System and Method

Abstract

The carrier system (100) provides one or more carriers (12) for carrying assay samples in an assay. The carriers are secured to a substrate (10) by a release layer (14). The carriers are suitable for receiving an assay sample, and the release layer is configured to release the carrier from the substrate in the presence of a biocompatible aqueous solution. To perform the assay, a biocompatible aqueous solution in which the assay sample is suspended is typically provided to the carrier system. The assay sample is received by the carrier, and the release layer is activated by the biocompatible aqueous solution to release the carrier.

Description

Technical Field

[0001] The present invention relates to carrier systems and methods, and in particular, to a carrier system for assays, a method for manufacturing a carrier system, and a method for performing an assay using a carrier system.

Background Art

[0002] An assay is an investigative procedure for qualitatively or quantitatively measuring the presence, amount, or activity of an analyte in an assay sample. Often, the assay sample is a cell or cell culture, and the analyte is a protein or gene sequence of that cell or cell culture, but more generally, the analyte can include metabolites, peptides, proteins, nucleic acids, extracellular vesicles, organelles, cells, or tissues.

[0003] Often, the assay sample is exposed to reagents prior to the measurement step. In fields such as biology and pharmacy, it is common for assay samples to be exposed to a number of different reagents. For example, if the analyte is a protein or gene sequence of a cell culture that serves as a disease model, different samples of the cell culture can be exposed to a number of different drug candidates. Typically, the interaction between the assay sample and the reagent takes place within a well plate. The measurement step may be performed within the well plate or elsewhere. In some cases, the measurement step may be performed, for example, under flowing conditions within a flow cytometer.

[0004] Some biological assay samples, such as certain types of cells, may be suspended in an aqueous solution. However, there are many biological assay samples that cannot be easily suspended in solution and are most viable when attached to a surface, such as adherent cells. Typically, the assay of such samples, including the steps of measurement, needs to be performed while the assay sample is attached to the bottom of a well plate. In such assays, the assay process can be disadvantageously limited by the fact that the assay sample attached to the bottom of the well plate cannot be easily transferred to another container without detaching it from the well plate. This can damage the sample.

[0005] Measuring an assay sample attached to a well plate is inefficient. Typically, the assay sample is attached or seeded across the entire well plate, but only 1 or 2% of the assay sample is actually imaged or measured. This is particularly problematic when the assay sample is a scarce resource, such as primary cells that are particularly difficult to culture or cells taken during a tumor biopsy.

[0006] Adherent cells are an example of an assay sample that is most viable when attached to a surface. For example, there is strong interest in assaying adherent cells under flowing conditions using a flow cytometer. However, this requires detaching the adherent cells from their attached state prior to the assay. This can bias the assay performed because the adherent cells may be damaged or the adherent cells forced into suspension may not accurately represent the normal biological state of the adherent cells. The significance of this problem is shown by the fact that in conventional assays, it is common to wait 12 to 48 hours after detaching the adherent cells from the substrate until the cells return to a normal or unperturbed state.

[0007] Another problem with performing an assay on an assay sample that is most viable when attached to a surface or tends to aggregate on a surface is that such an assay sample can tend to clog channels in a flow cytometry device. SUMMARY OF THE INVENTION

[0008] The present invention provides a carrier system for an assay, a method of manufacturing a carrier system for an assay, and a method of using a carrier system for an assay, as defined in the appended independent claims to which reference is hereby made. Preferred or advantageous features of the invention are set out in the dependent claims.

[0009] Thus, advantageously, a first aspect of the present invention may provide a carrier system for an assay, comprising a carrier or particle for carrying an assay sample. The carrier is fixed to a substrate by a release layer. Advantageously, the carrier is suitable for receiving an assay sample and the release layer is configured to release the carrier from the substrate in the presence of a biocompatible aqueous solution during use. For example, the release layer can be activated by a biocompatible aqueous solution to release the carrier. Thus, the release layer can release the carrier from the substrate when activated.

[0010] Generally, an assay sample for introduction into an array system can be suspended in a biocompatible aqueous solution. When a user of the carrier system wishes to perform an assay, contact between the biocompatible aqueous solutions carrying the assay sample can automatically release the carrier into the solution.

[0011] Thus, preferably, the carrier system contacts a biocompatible aqueous solution carrying an assay sample so that the assay sample (e.g., adherent cells) can attach to the carrier before the carrier is released into the solution and can carry the assay sample.

[0012] In a preferred embodiment, the assay sample may be adherent cells, and the carrier or particle may be sized such that the cells are suitable for adhering to the carrier. For example, as described in more detail below, the carrier may have a planar upper surface with a lateral dimension in the range of 5 to 200 micrometers that is suitable for adherent cells to attach to. The assay sample may then be carried via the assay on the carrier. In a particularly preferred embodiment, the carrier may be magnetic so that the carrier and the assay sample carried by the carrier can be oriented via the assay by the application of an external magnetic field.

[0013] Aspects of the invention are described herein with reference to carriers or particles immobilized on a substrate. In typical applications, the carrier is immobilized on the same substrate and may be, for example, one of a number of carriers or particles used to perform a multi-channel assay. Thus, in a typical embodiment of the invention, the substrate may carry many carriers, such as more than 10, 100, or 500 carriers, up to a number of 1000, 5000, or 10,000 or more. As described below, each carrier may be individually identifiable, or a subgroup of carriers may be identifiable, by a label such as a barcode carried by the carrier for use in a multi-channel assay. A plurality of carriers immobilized on a substrate, or assay sample carriers, may be referred to as a carrier array or particle array.

[0014] However, for the sake of simplicity, the embodiments of the invention described herein will typically be described with reference to only one carrier.

[0015] In a preferred embodiment, the assay sample may be introduced into a carrier system such that the assay sample can be received by or on the carrier. The assay sample may be a biochemical substance, a biological cell, or an organic sample. Preferably, the assay sample can be an adherent cell. The assay sample received on the carrier can preferably adhere, attach, seed, or bind to the surface of the carrier before the carrier is detached from the substrate. Then, the assay may be performed on the assay sample received on the detached carrier and supported by the detached carrier. The assay may include measuring the presence, amount, or activity of an analyte of the assay sample. When the assay sample is a cell, for example, the analyte may be a protein or gene sequence of the cell.

[0016] The assay sample received on the carrier can be convenient for manipulation and transportation. This can be advantageous when performing the assay, for example, to transfer the assay sample to a well plate, to expose the assay sample to a reagent, or to place the assay sample against a detector in the measurement step.

[0017] In a further embodiment of the present invention, for convenience, the reception of the assay sample on the carrier may be used to store the assay sample for use in future assays.

[0018] Conventional, prior art methods of storing cells for assays may include the following. - Detach adherent cells (e.g., chemically) and make a single cell suspension in a medium. - Concentrate the cells to about 1 M cells per mL (performed with a centrifuge). - Supplement the medium with 5 - 20% DMSO (dimethyl sulfoxide) or an equivalent amount. - Then, this mixture is added to a freezing tube (i.e., a cryogenic vial). - Next, the freezing tube is slowly cooled to -70°C at 1°C per minute (typically, this is done by immersing the vial in isopropanol and placing it in a -70°C freezer). - Then, the vial is stored at -70°C (in a freezer or in liquid nitrogen). - When thawing, the frozen vial is placed in a 37°C warm water bath until thawed. - Next, fresh medium is added, the cells are spin - down, and fresh medium is added again. - Then, the cells are seeded into a flask or plate or dish and the assay is performed.

[0019] Using one embodiment of the present invention, this method can be significantly improved as follows. - As described herein, adherent cells are attached to a carrier attached to a substrate. - Optionally, as described herein, the carrier is detached from the substrate into the liquid. Alternatively, the carrier may be retained on the substrate. - Then, the cells are either cultured for 1 - 2 days or directly frozen (the former is preferred to maintain healthy cells). - The substrate carrying the carrier, i.e., the free carrier carrying or supporting the cells, is in a medium refreshed with a medium containing 5 - 20% DMSO, and it is confirmed that there are approximately 1 M cells per 1 mL of liquid. When the medium is refreshed, either a centrifuge or gravity may be used to concentrate the carrier. Alternatively, when a magnetic carrier is used as described herein, the carrier may be magnetically pulled down or retained when the medium is refreshed. - Then, the protocol will be carried out in the same manner as conventional cell freezing, except that the cells are received on a carrier ready for use in the assay.

[0020] Embodiments of the present invention provide many advantages. - The cells do not need to be detached before freezing. - After freezing, the cells do not need to be seeded or attached to any substrate. - Viable, adherent cells can be preserved. - This is useful when researchers want to perform experiments (requiring adherent cells) but also want to save some assayed samples for later analysis or additional experiments. - This is useful when people want to culture a large batch of cells and perform multiple experiments over a long period of time. Then, the entire batch or multiple cells can be attached to a carrier and then frozen. Then, when people want to perform an experiment, a portion or all of the cells can be thawed. This ensures minimal variability and bias between experiments. Conventionally, what people have to do is to continuously culture the cells (through multiple passages, i.e., the cycle of detaching and reseeding when the cells grow too much during culture), but mutations and changes accumulate over these different passage steps. Another known solution is to always thaw a new batch before the experiment. However, this is not only labor-intensive (as thawing, 2-day culture, detachment, and reseeding are required), but also introduces variability and potentially bias. - Embodiments of the present invention are useful when people want to perform rapid assays. This approach ensures that people do not need to perform the "thaw, 2-day culture, detachment, reseeding" steps before the assay. In this situation, the cells are thawed and the experiment / assay can be started directly (without the detachment and reseeding steps) or after 1-2 days of culture.

[0021] Conventionally, assay-ready cells are available, but these cells are still suspended in a liquid and need to be attached to a substrate before the assay. Embodiments of the present invention ensure that these assay-ready cells are all frozen in the same "state" (e.g., how many passages the assay-ready cells have undergone, which cell cycle step the assay-ready cells are in, confirming that there are no artifacts such as mutations or metabolic problems in the assay-ready cells). Thus, in one embodiment of the present invention, the assay-ready cells on the carrier may be sold in a usable state, for example, in a frozen state. Further, as described herein, the assay-ready cells may be on a carrier that is individually identifiable, for example, by a barcode or other readable label on each carrier.

[0022] In a preferred embodiment of the carrier system of the present invention, the release layer may be activatable in the presence of an assay sample solution or assay sample medium for containing or supporting the assay sample. For example, the assay sample solution may contain a biocompatible aqueous solution and the assay sample. Thus, a single act of introducing the assay sample solution into the carrier system can result in the assay sample being received by the carrier and the carrier being detached from the substrate.

[0023] Many assay samples need to be maintained and transported in a biocompatible aqueous solution to maintain viability. Thus, the provision of a carrier for receiving an assay sample, which is fixed to a substrate by a release layer configured to detach the carrier in the presence of a biocompatible aqueous solution, is particularly convenient. For example, the biocompatible aqueous solution may be non-cytotoxic.

[0024] In a preferred embodiment, the release layer may be configured such that after activation of the release layer, the biocompatible aqueous solution maintains biocompatibility. Preferably, the release layer may not release non-biocompatible or toxic components into the biocompatible aqueous solution. Otherwise, such components may render the biocompatible aqueous solution toxic to the assay sample. More generally, preferably, the release layer may not release any material or component that can modify or change the assay sample in any way into the biocompatible aqueous solution. Advantageously, such a release layer may not affect the results of an assay performed using a carrier system. For example, the release layer may be formed of a biocompatible material.

[0025] In a preferred embodiment, the assay sample is the most viable when received on a surface. An example of such an assay sample may be adherent cells. When such an assay sample is received by a carrier or on the surface of a carrier and the carrier is detached from the substrate, the assay sample may remain on the carrier rather than move to another surface. This may mean that such an assay sample received on the detached carrier and carried by the detached carrier is convenient for manipulation and transportation. Furthermore, such an assay sample may remain in a representative biological state during the performance of the assay. This may reduce any bias in the assay of such an assay sample. In particular, there may be no need to force suspension of such an assay sample through mechanical or chemical means as required in prior art methods. Advantageously, aggregation of such assay samples may also be reduced.

[0026] Advantageously, an assay sample received on a carrier embodying the present invention may be measured using, for example, a flow cytometer or fluorescence microscopy.

[0027] In a preferred embodiment, the release layer may comprise a water-activatable material, that is to say, a material that releases the carrier or particles in the presence of water, such as water in a biocompatible aqueous solution. The water-activatable material may be a water-soluble material. The water-soluble material may dissolve in the presence of water and release the carrier. The release layer may dissolve completely or may not dissolve completely. The release layer may dissolve only to such an extent that the bond between the release layer and the carrier is weakened and the carrier is released from the substrate.

[0028] Alternatively or additionally, the release layer may comprise a material having other properties that change in the presence of water or an aqueous solution. For example, the adhesiveness of the material of the release layer may be reduced in the presence of water or an aqueous solution. Similar to the water-soluble release layer, these release layers may allow the carrier to be released from the substrate in the presence of water, but preferably have the advantage that any component of the release layer can be released without adding it to a solution that can affect the assay sample.

[0029] As will be described in more detail later in this document, in a preferred embodiment, the manufacture of the carrier system may include a lithography process. The release layer may then be formed of a material suitable for or compatible with the required lithography process. The release layer may be applied to the substrate prior to at least some steps of the lithography process, and then the carrier may be formed on the release layer. Advantageously, the release layer may not be affected by the processes and chemicals used in the lithography process in order to be suitable for the lithography process.

[0030] In a preferred embodiment, the release layer may comprise a material that is not activatable in a non-aqueous solvent such as ethanol. As will be described in more detail later in this document, a method of manufacturing a carrier system may include applying a coating adapted to receive an assay sample to the carrier. Advantageously, this process may be carried out while the carrier is fixed to the substrate by the release layer. For example, the coating may be applied by immersing the carrier system in a solution for a time such as between 10 and 120 minutes to form or deposit the coating, and the coating may be a polymer. Generally, such a solution contains a non-aqueous solvent such as ethanol, so advantageously, the release layer may be unaffected or not activated by such a solvent so that it can hold the carrier fixed to the substrate while the carrier system is exposed to the non-aqueous solvent. In particular, advantageously, the release layer may not release the carrier when exposed to a non-aqueous solvent during the time and under the conditions required for the manufacture of the carrier.

[0031] Alternatively or additionally, the carrier system may be subjected to a sterilization process while the carrier is fixed to the substrate by the release layer. Sterilization may be part of the manufacturing process or the preparation of the carrier system for performing an assay. Advantageously, sterilization eliminates unwanted types of living organisms or biological agents that may otherwise interfere with the assay performed using the carrier system. Sterilization may include immersing the carrier system in a non-aqueous sterilizing solution such as ethanol. The sterilizing solution may contain pure ethanol. The carrier system may be immersed in the sterilizing solution for up to 10, 15, 20 or 25 minutes, typically up to about 30 minutes or 1 hour. If the release layer comprises a material that is not soluble in a non-aqueous solvent such as ethanol, advantageously, the release layer may hold the carrier fixed to the substrate while the carrier system is immersed in the non-aqueous solvent. In particular, advantageously, the release layer may not release the carrier when exposed to a non-aqueous solvent during the time and under the conditions required for sterilization of the carrier.

[0032] The release layer may contain sugar. Sugars are examples of materials that can be biocompatible. The release layer containing sugar may release the carrier in the presence of a biocompatible aqueous solution such as an aqueous solution. The sugar may be water-soluble. Preferably, the release layer may contain a sugar such as dextran. The release layer containing dextran may be biocompatible. In the manufacture of the carrier system, the release layer may be applied to the substrate using a spin coating method. Advantageously, the release layer containing sugar, especially dextran, may be suitable for spin coating.

[0033] Typically, dextran may contain polymer molecules having a length in the range of about 3 to 2000 kDa or more. Any solution of dextran may be spin-coated to form a release layer. Dextran of 70 kDa has been found to provide a release layer activation time between 1 and 10 minutes. Larger dextran molecules in the range of 5000 to 40000 kDa may be used to provide lower solubility in biocompatible aqueous solutions and longer release times. Thus, smaller dextran molecules in the range of 3 kDa or 50 kDa to 500 kDa or 2000 kDa may be used to provide shorter release times, and larger dextrans such as in the range of 2000 kDa or 3000 kDa or 5000 kDa to 6000 kDa or 10000 kDa or 40000 kDa may be used to provide longer release times.

[0034] The release layer containing sugar, such as dextran, may be suitable for a lithography process and may not be activatable in a non-aqueous solvent such as a solvent containing ethanol or in pure ethanol.

[0035] The parameters of the release layer, such as its material, structure, and thickness, may be selected or designed according to the desired time required for the carrier to be released after the introduction of the biocompatible aqueous solution into the carrier system. In a preferred embodiment, the parameters of the release layer may be selected to release the carrier after a time when an assay sample may have been received on the carrier. Receiving an assay sample on the carrier while the carrier is in contact with the substrate may ensure that the assay sample is received only by the exposed surface of the carrier. Typically, since only one surface of the carrier can be measured during the assay, advantageously, this may ensure that most of the assay sample is measured, reducing or eliminating waste of the assay sample. This is particularly advantageous when the assay sample is a scarce resource, such as primary cells that are particularly difficult to culture or cells collected during a biopsy.

[0036] A release layer containing a sugar such as dextran may release the carrier relatively rapidly, such as in 5 or 10 seconds or less, in the presence of a biocompatible aqueous solution. Typically, some assay samples may require much longer than 5 seconds to be received on the carrier after introduction of the assay sample into the carrier system. For example, adherent cells may require more than 3 hours to be received on the carrier. Thus, the release layer may include a material having a longer release time.

[0037] The release layer may include polyvinyl alcohol (PVA). The release time of a release layer containing polyvinyl alcohol may be much longer than that of a release layer containing dextran. A release layer containing polyvinyl alcohol may be configured to release the carrier about 1, 2, 3, 6, or 9 hours, up to 12 hours or more, after the introduction of the biocompatible aqueous solution and the assay sample into the carrier system. Thus, a release layer containing polyvinyl alcohol may be designed to release the carrier at an appropriate time after the introduction of the biocompatible aqueous solution so that an assay sample, such as adherent cells, is received on the carrier.

[0038] Similar to sugars, polyvinyl alcohol is a material that can be considered biocompatible. The release layer containing polyvinyl alcohol may be water-soluble, may be suitable for spin coating, and may be compatible with the lithography process. The release layer containing polyvinyl alcohol may not be activatable in a non-aqueous solvent such as a solvent containing ethanol or in pure ethanol.

[0039] Other materials may be used to form the release layer, such as PLGA (PLG or poly(lactic-co-glycolic acid)) or poly(acrylic acid) (PAA). The structure of the release layer may also be formed from multiple materials such as polymers and / or sugars deposited layer by layer. As described above, manufacturing a carrier system embodying the present invention may include the step of applying a coating adapted to receive an assay sample to the carrier. Alternatively or additionally, the carrier system may be subjected to sterilization. As described above, each of these processes may be carried out using a non-aqueous solvent such as ethanol, and advantageously, the carrier may remain fixed to the substrate in the presence of the non-aqueous solvent.

[0040] Alternatively, in some embodiments, the material, structure, and thickness of the release layer may be selected to allow the use of an aqueous solution during manufacturing, for example, in the application of the coating or during the sterilization process. If the parameters of the release layer are selected such that the carrier is only released after a time longer than the cumulative time the carrier system is immersed in the aqueous solution for any or both of the above processes, these processes may be carried out and the carrier may remain fixed to the substrate. Also, it should be noted that the release layer should still provide the time for the assay sample to be received on the carrier while the carrier is fixed to the substrate by the release layer when the assay is to be performed.

[0041] Generally, to achieve the desired release time, the material of the release layer and other properties such as thickness and structure may be varied or predetermined.

[0042] In a preferred embodiment, the carrier may comprise a magnetic material. This can have many advantages in the assay. When an external magnetic field is applied, the carrier has a sufficient magnetic moment for the external field and a desired force may be applied to the carrier.

[0043] For example, even after the release layer has released the carrier, the force may be applied and the carrier in contact with the substrate may be held. As described above, it can be advantageous for an assay sample to be received by the carrier while the carrier is in contact with the substrate. Even after the release layer has released the carrier, by applying an external field and holding the carrier in contact with the substrate, the carrier may be held at a predetermined position on the substrate for any length of time. For example, this may eliminate the need to match the release time of the release layer to the typical time required for a particular assay sample to be received by the carrier, or allow a carrier held by a release layer designed to have a particular release time to receive an assay sample that requires any length of time longer than the release time received on the carrier. In other words, a release layer having an activation time shorter than the typical time required for a particular assay sample to be received by the carrier may be used.

[0044] Similarly, advantageously, the force applied by the external field is sufficient to move or drive the carrier through the solution during the assay while carrying the assay sample. For example, if the sample is a cell, the force should be sufficient to move the carrier and the cell.

[0045] The carrier may be defined or fabricated by lithography. Advantageously, a carrier defined by lithography can have a high aspect ratio. Advantageously, such a carrier can have a large surface area for receiving an assay sample relative to the volume of the carrier. In a preferred embodiment, for convenience, the carrier may be defined or fabricated by lithography on or above the release layer of the carrier system.

[0046] The carrier may include a photoresist layer that can be an artifact of a lithography process used to define or fabricate the carrier. Advantageously, the photoresist layer can include one or more materials that are not water-soluble.

[0047] During the manufacture of the carrier, the photoresist layer may be patterned by exposing it to radiation and washing away or dissolving regions of the photoresist layer with a solvent. Typically, in a lithography process, the photoresist is water-soluble and the solvent is an aqueous solvent. In embodiments of the present invention, a water-insoluble photoresist is preferred. Then, advantageously, the step of washing away the photoresist may include a solvent other than an aqueous solvent. Thus, advantageously, the release layer may not be affected by the washing process.

[0048] The photoresist layer may include an SU-8 photoresist. The SU-8 photoresist may be soluble in a solvent containing, for example, 2-methoxy-1-methylethyl acetate, γ-butyrolactone or cyclopentanone. Advantageously, the SU-8 photoresist may not be soluble in a biocompatible aqueous solution. Thus, the SU-8 photoresist may be biocompatible if present in an assay.

[0049] In a preferred embodiment, the surface of the carrier may be adapted or modified to receive an assay sample. Advantageously, such adaptation can improve the efficiency or rate at which the assay sample is received by the surface, thus shortening the time for the assay sample to be received on the carrier and, advantageously, increasing the strength of the bond or attractive force between the assay sample and the carrier so that the assay sample is securely bound to the carrier. This can also reduce any tendency for the assay sample to move or separate from the carrier during the assay. The surface of the carrier may be adapted to receive adherent cells. The adapted surface may be the surface of the carrier that is exposed when the carrier is fixed to a substrate.

[0050] The surface of the carrier may include a coating adapted to receive an assay sample. A carrier including such a coating may be referred to as being biofunctionalized. The coating may be adapted to receive adherent cells as an assay sample. The coating may be a charged coating. This may be particularly preferred when the assay sample is originally charged. For example, typically, cells have a membrane potential between minus 40 millivolts and minus 80 millivolts. Providing a coating that defines a positively charged surface may facilitate the attachment of the assay sample on the charged surface of the carrier. Advantageously, it has been found that providing a charged surface, preferably a positively charged surface, can lead to more effective cell adhesion. The coating may include a charged polymer.

[0051] In some embodiments, the coating is applied onto the gold layer of the carrier in the form of a gold cap.

[0052] The gold cap of the carrier may be present only on one side or surface of the carrier. In particular, the gold cap may be present only on the surface of the carrier that is exposed while the carrier is fixed to a substrate. This ensures that only one surface of the carrier is biofunctionalized and thus only one surface of the carrier tends to receive an assay sample. The advantages of receiving an assay sample on only one surface of the carrier are described herein. In some embodiments, the carrier may be fabricated using a lithography process. The lithography process may be particularly suitable for fabricating a carrier having only a specific surface including a gold cap.

[0053] The polymer (charged polymer) may be covalently attached to the gold cap. The surface adapted to receive the assay sample may include a gold cap layer to which the polymer is covalently attached by a thiol group. Advantageously, this ensures that each polymer adsorbed on the surface has the same orientation. Alternatively, the polymer may be adsorbed to the carrier's gold cap by van der Waals forces.

[0054] The charged polymer may be a polymer containing a positive charge transport group. The polymer may be polyornithine or poly-d-lysine. The polymer may be a polyelectrolyte.

[0055] Alternatively or additionally, the coating may include a plurality of ligands. The plurality of ligands may include antibodies. When the assay sample of the assay is a cell, the plurality of ligands may include antibodies that specifically bind to cell receptors such as integrins.

[0056] When the assay sample is a cell, alternatively or additionally, the coating may include an extracellular matrix protein. The extracellular matrix protein may be a protein selected to increase or enhance cell adhesion. The protein may be collagen, laminin, or Matrigel, a protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.

[0057] The surface of the carrier may include a physical structure adapted to receive adherent cells. The physical structure or topography of the surface can affect the degree to which adherent cells adhere or seed on the surface. Advantageously, cell adhesion can be increased by increasing the surface area of the surface of the carrier. The surface area may be increased by increasing the roughness of the surface. For example, the surface may be etched using an ion beam.

[0058] The surface hardness can also affect the rate or efficiency at which adherent cells are received on that surface of the carrier. Cell adhesion is closely related to the Young's modulus of the surface. Therefore, the hardness of the surface of the carrier, i.e., the Young's modulus, may be adjusted to optimize the adhesion of the cells, which are the assay samples.

[0059] In summary, in one example where the assay sample contains adherent cells, the steps in an assay according to a typical embodiment of the present invention may include the following. 1. The carrier array is placed in a well plate, flask, or any other cell culture vessel. 2. The detached cells (single cells) are mixed with the medium and loaded onto the carrier array. 3. The cells will settle and attach to the carrier (typically, at least 2 - 3 hours). 4. The aqueous medium slowly dissolves the sacrificial layer (release layer). 5. The carrier with the adherent cells is detached into the liquid. 6. The carrier with the cells can be moved, assayed, measured, stored, etc.

[0060] As described above, the carrier may include a magnetic material. When an external field is applied, the carrier may have a magnetic moment sufficient for the external field to apply a desired force to the carrier. The desired force may depend on the application of the carrier. In a practical implementation, typically, the applied external field is less than 2T, or less than 1T or 0.5T, and may typically be greater than 0.05T, or 0.1T or 0.25T. A carrier containing a magnetic material may be movable by the application of an external magnetic field. For example, the carrier may be movable within a liquid medium. The liquid medium may be a biocompatible aqueous solution introduced into the carrier system to detach the carrier. The magnetic moment may be due to the magnetization of the carrier itself or may be induced in the carrier by an external field. However, it is important that the carrier contains sufficient magnetic material to generate the desired force. The magnetic moment that can be generated by the external field applied to the carrier may depend on the total volume V of the magnetic material in the carrier multiplied by the magnetization Ms of the material. Therefore, preferably, the value of V.Ms of the carrier embodying the present invention is greater than a predetermined value such as 10 -18 J / T or 5x10 -18 J / T or 10 -17 J / T or greater than a predetermined value such as.

[0061] For example, the magnetic material may include a material selected from metals or metal alloys such as Fe, Co, Ni, CoFe, CoFeB, FePt, CoNi, NiFe, etc.

[0062] The external field may be applied to orient the carrier and the assay sample received on the carrier with respect to the detector. Advantageously, this may improve the consistency and quality of the results from an assay performed using such a carrier when the carrier can be placed in an optimal position and angle with respect to the detector. The ability to orient the assay sample received on the carrier is particularly advantageous when the carrier is in a suspended state in, for example, an aqueous solution and may be particularly beneficial in microfluidic or flow cytometry applications.

[0063] The structure of the magnetic carrier enables many advantages in assays, such as changing the liquid in which the carrier and the assay sample are supported, selecting a specific carrier (e.g., after measurement for collection), and enabling the sedimentation of the carrier to the bottom of the well or flask with the surface of the carrier carrying the assay sample facing up (so that the assay sample is not pushed between the carrier and the well).

[0064] Next, a preferred embodiment of an imaging assay using a magnetic carrier may include the following steps with respect to an assay sample containing cells. - The assay sample adheres to the carrier, and after the release layer is activated, a magnetic field is used to hold the carrier on the substrate. - The assay sample (on the carrier) is transferred to a new container or well (to remove non - adherent cells (i.e., any cells not adhering to the carrier) and refresh the medium). - With the cells facing up (for incubation or cell growth), the carrier with the cells is magnetically pulled down to the bottom of the container. - Add assay reagents (e.g., drugs). - Remove the liquid (using a magnetic field to fix the carrier in a predetermined position). - Sequentially add various assay liquids (e.g., wash buffer, labeled antibody) (using a magnetic field to fix the carrier in a predetermined position when the liquid is exchanged). - Invert the carrier (using a magnetic field) and perform microscopic imaging through the bottom of the well.

[0065] In an alternative embodiment, the use of the carrier embodying the present invention using a flow - based assay (although described repeatedly in the context of an assay sample containing cells) can provide additional advantages such as enabling imaging of groups of cells or single cells on the carrier that do not aggregate (clog) and pass through the detector, and enabling measurement using either fluorescence intensity (spectrophotometry) or taking a full image of the cells on the carrier.

[0066] In one embodiment of the present invention, the carrier or particle may include a layer structure between the upper surface of the carrier and the opposing bottom surface of the carrier, the layer including one or more magnetization layers, preferably, the ratio of the lateral dimension of one or more magnetization layers to the thickness or collective thickness of one or more magnetization layers is greater than 500. A carrier having such a structure may have a low stray magnetic field at the surface of the carrier. Advantageously, this may mean that when a plurality of carriers having the same structure are provided, the carriers may not interact with another carrier. In particular, the carriers may not aggregate or form lumps. Such cases may occur regardless of whether the carrier has remanent magnetization or not.

[0067] The carrier may further include a non-magnetic layer, advantageously, the non-magnetic layer may provide mechanical support to the magnetic layer and may determine the physical properties of the carrier such as its mechanical properties and its density. The non-magnetic layer may provide a suitable substrate for the magnetization layer. Thus, advantageously, the non-magnetic layer may include materials selected from Al, Ta, Pt, Pd, Ru, Au, Cu, W, MgO, Cr, Ti, Si, Ir, SiO2, SiO, Sn, Ag, polymers and plastics, alloys of these materials, as well as composites or mixtures containing these materials.

[0068] In a preferred embodiment, the carrier may include readable information such as a barcode or a readable code selected from two-dimensional codes. This may enable the carrier to be remotely identified, for example, by reading the information using a camera and appropriate software. A multi-channel assay may be implemented by providing a plurality of carriers each carrying readable information.

[0069] In a preferred embodiment, the readable information may be used to identify an assay sample received on a carrier. In a multiplex assay, two or more types of assay samples may be analyzed at once. If the assay sample is a cell, two or more different types of cells may be analyzed at once. The readable code may indicate the type of assay sample on a particular carrier. The assay may be performed using a plurality of different coded carriers, and the different coded carriers may carry different types of assay samples. After the assay sample is received on the carrier, carriers having different types of assay samples may be pooled. For example, a plurality of carriers, each containing (carrying) a different type of cell, may be pooled or mixed in a well plate containing a particular drug in order to expose the various cells to the drug. Advantageously, the identification of carriers using readable information in this manner may provide a multiplex platform by which carriers, and thus assay samples received on the carriers, can be accurately distinguished from one another.

[0070] A particular type of assay sample may be received by a carrier having the same readable information. In that case, the readable information only needs to distinguish carriers carrying different types of assay samples. In other words, the number of assay channels, i.e., the plex, is equal to the number of types of individual assay samples used in the assay. However, it may be advantageous to provide each carrier with unique readable information. A subset of carriers may include unique readable information related to a particular type of assay sample. The unique readable information of the subset of carriers may also be used to identify test conditions experienced by each assay sample in the subset during the assay, e.g., a particular drug to which the assay sample is exposed during the assay. In this case, the readable information needs to be able to distinguish all carriers of the plurality of carriers of the carrier system. In other words, the plex of the assay may be equal to the number of carriers used in the assay.

[0071] The use of barcodes or two-dimensional codes can provide a significantly more robust process for distinguishing different carriers with minimal crosstalk between plex channels than existing multiplex assay platforms. Further, the use of readable information in such a way can enable the use of many more multiplex channels than are currently possible. For example, barcoding or two-dimensional codes can enable 1000 plex, or 10,000 plex, or more as needed (i.e., the assay may include 1000, or 10,000, or more multiplex channels). The use of barcodes or two-dimensional codes can be particularly advantageous when each carrier is required to contain unique readable information.

[0072] In a preferred embodiment, the carrier (or each carrier) may include a magnetic material, and an appropriate external magnetic field may be applied to induce, move, or drive the carrier through a liquid medium to a predetermined position for reading the code or information. The liquid medium may include or be composed of an aqueous solution introduced into the carrier system to detach the carrier. For example, a carrier having a high aspect ratio shape with a large top or bottom surface for displaying the code or information may be oriented to be in contact with a substrate or other support surface for easy reading of the code or information. A magnetic field may be applied to induce the carrier to the reading position, and the assay result may be obtained by reading the readable code and the assay sample received on the carrier.

[0073] In a preferred embodiment, the carrier system may include a plurality of carriers for receiving assay samples fixed to the substrate by a release layer. The assay samples may be received on each carrier. The same type of assay sample may be received on each carrier. Alternatively, different types of assay samples may be received on some or each of the carriers. By providing a plurality of carriers, advantageously, a multiplex assay may be performed using the carrier system.

[0074] A second aspect of the present invention can provide a method for manufacturing a carrier system for an assay, the method including the steps of providing a substrate, forming a release layer on the substrate, and depositing or forming a carrier on the release layer such that the carrier is fixed to the substrate, wherein the release layer is configured to detach the carrier from the substrate in the presence of a biocompatible aqueous solution during use.

[0075] In a preferred embodiment, the step of forming a release layer on a substrate may include spin-coating the release layer. The release layer may include a material particularly suitable for spin-coating. For example, the release layer may include a sugar such as dextran, or polyvinyl alcohol (PVA), or poly(lactic-co-glycolic acid) (PLGA), or poly(acrylic acid) (PAA).

[0076] In a preferred embodiment, the step of depositing a carrier on the release layer may include creating a carrier on the release layer. In other words, the method of manufacturing a carrier system may include the step of creating a carrier. For convenience, this may enable the carrier to be manufactured in the same process as the manufacture of the carrier system. Alternatively, the step of depositing a carrier may include depositing a carrier already created on the release layer. For example, the carrier may be manufactured by a third party.

[0077] A plurality of carriers may be deposited or created on the release layer.

[0078] A method of manufacturing a carrier system may include a lithography process. In particular, the carrier may be defined by lithography. Advantageously, this may provide carriers with a high aspect ratio. Fabrication of the carrier may include a lithography process. The lithography process may include a physical vapor deposition process, and advantageously, the physical vapor deposition process enables sub-nanometer control in the deposition of various layers used to fabricate the carrier. The thickness of the carrier fabricated using the lithography process may be on the order of nanometers, and the minimum size of the carrier in the lateral dimension may be between 5 and 200 microns, or alternatively between 20 and 200 microns. The carrier (in the lateral dimension) may be of any convenient shape, such as square or rectangular or circular. Carriers having a minimum lateral dimension of 5 or 10 or 20 microns may be suitable for receiving a single cell and thus may be advantageous in assays where a single cell is the target. Larger carriers, for example, carriers having a minimum lateral dimension close to 200 microns, may be suitable for receiving multiple cells. In a preferred embodiment, the carrier may have a minimum lateral dimension of 100 microns.

[0079] Fabrication of the carrier may include forming a photoresist layer on or over a release layer.

[0080] The photoresist layer may be spin-coated over the release layer. As described above, advantageously, the photoresist layer may include one or more materials that are not water-soluble, so portions of the photoresist layer may be washed away or dissolved using a non-aqueous solvent that does not affect the release layer.

[0081] If the photoresist layer is not soluble in an aqueous solvent, it may be considered biocompatible. For example, the photoresist layer may include a biocompatible polymer.

[0082] A suitable photoresist layer may be a SU-8 photoresist layer. The SU-8 photoresist layer may not be water-soluble. The lithography process may include applying a photomask to the photoresist and exposing the photoresist layer to ultraviolet light before washing away or dissolving the unnecessary areas of the photoresist layer.

[0083] When the photoresist layer is patterned to define the regions of the carriers on the substrate, the release layer between the carriers may be removed, if necessary. Advantageously, this may reduce the volume of the release layer exposed to the biocompatible aqueous solution during subsequent release of the carriers, reduce any concentration of the release layer that dissolves in the solution, and minimize any effect that concentration may have on the assay results.

[0084] As described herein, during fabrication of the carriers, layers of subsequent materials may be deposited on the carriers. These materials may also be deposited on the substrate between the carriers or on the release layer. If any such material is deposited between the carriers, it is important that it does not prevent access of the biocompatible aqueous solution to the edges of the release layer under the carriers when the assay is performed. If necessary, the material between the carriers may be removed during fabrication to ensure that the edges of the release layer under the carriers are exposed so that the release layer can be activated by the biocompatible aqueous solution to release the carriers.

[0085] A method for manufacturing a carrier system may further include the step of sterilizing the carrier system by immersing the carrier system in ethanol after depositing, or forming, or fabricating a carrier on a release layer. Advantageously, sterilization can eliminate unwanted types of living organisms or biological agents that can conversely interfere with an assay performed using the carrier system. Typically, the carrier system can be immersed in the sterilizing solution for up to 10, 15, 20, or 25 minutes. Sterilization may include immersing the carrier system in a non-aqueous sterilizing solution such as ethanol. The sterilizing solution may include pure ethanol. This can be advantageous when the release layer includes a material such as dextran or polyvinyl alcohol that cannot be activated in a non-aqueous solvent since the carrier remains fixed to the substrate while the carrier is immersed in the sterilizing solution. Even if the sterilizing solution affects the release layer, a similar effect can be achieved if the release time of the release layer is longer than the time the carrier system is immersed in the sterilizing solution.

[0086] The step of sterilizing the carrier system may also be performed using a dry heat sterilization method, a steam sterilization method, or a gas sterilization method. Dry heat sterilization may be most suitable for this step since the release layer is least affected by this sterilization method.

[0087] The step of depositing, or fabricating, a carrier may include forming a magnetic structure or layer. In particular, when the method includes a lithography process, the method may include depositing a magnetic material on a photoresist layer. Further materials such as gold may also be deposited on the photoresist layer or on the magnetic material of the carrier. A gold cap may provide biocompatibility. Additionally, a barcode, a QR code, or other readable information may be added to the carrier by lithography.

[0088] The method may further include depositing a gold layer on the carrier and forming a cap. Advantageously, the gold cap may be biocompatible. The gold may be deposited only on the surface of the carrier that is exposed when the carrier is in contact with the substrate.

[0089] The method may further include adapting the surface of the carrier to receive an assay sample. In a preferred embodiment, the surface of the carrier may be adapted to receive adherent cells.

[0090] The step of adapting the surface of the carrier to receive an assay sample may be performed before the step of depositing the carrier on the release layer. Alternatively, the step of adapting the surface of the carrier to receive an assay sample may be performed after the step of depositing the carrier on the release layer. When the step of depositing the carrier on the release layer includes creating the carrier on the release layer, the step of adapting the surface of the carrier to receive an assay sample may be a step of that creation process.

[0091] As described above, the step of adapting the surface of the carrier to receive an assay sample may include applying a coating to the surface of the carrier.

[0092] The coating may be a charged coating. The coating may include a charged polymer. The charged polymer may be a polymer containing a positive charge transport group. The polymer may be a polyelectrolyte. The charged polymer may be a polymer containing a positive charge transport group. The polymer may be polyornithine or poly-d-lysine.

[0093] The polymer may be applied by immersing the carrier system in a solution containing the polymer for a predetermined time, such as between 20 and 120 minutes. The solution may contain the polymer at a concentration between about 1 nM and 10 mM, depending on the polymer used. The solution may contain a non-aqueous solvent, such as ethanol. The solution may consist of a non-aqueous solvent and the polymer. The use of a non-aqueous solvent may be advantageous when the release layer contains materials such as dextran or polyvinyl alcohol that may not be activatable in a non-aqueous solvent. This may be because the carrier remains fixed to the substrate while the carrier is immersed in the solution containing the polymer.

[0094] The polymer may contain a thiol group, which may be a thiol group that covalently bonds to the gold cap of the carrier. Advantageously, this ensures that each polymer bound on the surface has the same orientation. Alternatively, the polymer may be adsorbed to the gold cap of the carrier via van der Waals forces.

[0095] Alternatively or additionally, the coating may contain a plurality of ligands. The plurality of ligands may include antibodies. The plurality of ligands may include antibodies that specifically bind to cell receptors such as integrins. Alternatively or additionally, the coating may contain an extracellular matrix protein. The extracellular matrix protein may be a protein selected to increase or enhance cell adhesion. The protein may be collagen, laminin, or Matrigel, a protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.

[0096] The step of adapting the surface of the carrier to receive the assay sample may include changing the surface topology of the carrier, for example, by increasing the surface area of the carrier. The surface area may be increased by increasing the roughness of the surface. For example, the surface may be etched using an ion beam. Alternatively or additionally, dots, pits, protrusions, or grooves may be provided on the surface of the carrier.

[0097] The step of adapting the surface of the carrier to receive the assay sample may include changing the hardness of the surface of the carrier. This may be achieved by applying a coating comprising a dense layer of protein or polymer.

[0098] A third aspect of the present invention may provide a method of performing an assay using a carrier system such as that of the first aspect of the present invention, or a manufactured carrier system such as that of the second aspect of the present invention described above. The method includes introducing a biocompatible aqueous solution into the carrier system to detach the carrier. The method may further include introducing an assay sample onto the carrier such that the assay sample is received by the carrier. Advantageously, the assay sample may be received by the carrier while the carrier is in contact with the substrate. The assay may be performed on the carrier having the assay sample received thereon. The advantages of the assay sample received on the carrier while the carrier is in contact with the substrate are described in connection with the first aspect.

[0099] In a preferred embodiment, the carrier may comprise a magnetic material, and the method may further comprise applying a magnetic field to the carrier, the magnetic field acting to hold the carrier in contact with the substrate even after the release layer has released the carrier from the substrate. This can be particularly advantageous when the release layer releases the carrier in a time shorter than the typical time for an assay sample to be received by the carrier. For example, if the release layer contains a sugar such as dextran, in the presence of a biocompatible aqueous solution, the release layer may release the carrier in 5 seconds or less. However, the typical time for an assay sample to be received by the carrier can be longer than 5 seconds. Advantageously, then, during the time required for the assay sample to be received by the carrier, while the magnetic field holds the carrier in contact with the substrate (by magnetically applying the force pressing the carrier against the substrate), the assay sample can be received by the carrier. This can eliminate the need to match the release time of the release layer to the typical time required for a particular assay sample to be received by the carrier while maintaining the advantage of receiving the assay sample on the carrier while the carrier is in contact with the substrate.

[0100] The magnetic field may be applied to hold the carrier in contact with the substrate for at least 5 seconds. In other words, the magnetic field may be applied to hold the carrier in contact with the substrate for a time longer than the typical time for activating a release layer containing a sugar such as dextran in the presence of water. The magnetic field may be applied for much longer than 5 seconds. For example, the magnetic field may be applied for at least 1, 5 or 10 minutes, or at least 1, 3 or 12 hours. In a preferred embodiment, the magnetic field may be applied for 2 to 4 hours, or about 3 hours, when the assay sample is adherent cells. This is the typical time for adherent cells to be received on the carrier. Typically, the magnetic field may be applied for less than 10 minutes, or less than 1, 3, or 12 hours, but this time will depend on the assay application.

[0101] It is clear that the features described in connection with one aspect of the invention may be applicable to other aspects of the invention.

[0102] Carriers or carrier systems containing carriers for receiving assay samples have been described above. Particularly preferred features regarding the carriers have also been described above. However, it should be understood that the carrier system may include any carrier suitable for receiving and carrying a sample that can be fixed to a substrate by a release layer, and such a carrier system may have the advantages described above.

[0103] A further particular preferred embodiment of the present invention may include carriers on a wafer or on a substrate, with or without pre-functionalization. The wafer may carry carriers in the range of 1000 - 10 million, or more carriers, depending on the customer's needs. Alternatively, the carriers on the wafer or on the substrate may be provided with each wafer in a state where it is placed in the wells of a well plate. In this case, typically, depending on the size of the wells, there may be 100 - 10,000 carriers per wafer. An example of this embodiment may be a well plate containing carriers and a substrate already installed in one or more wells. In a further embodiment, the carriers on the wafer or on the substrate may be provided in a state where assay samples such as cells are already attached in a frozen state and ready for assay. Typically, such a wafer may carry 1000 - 100,000 carriers.

[0104] The carrier system embodying the present invention may be provided to the user in various forms. In one embodiment, a carrier system containing carriers on a substrate may be sterilized and packaged, for example, in a dry, sterile package or wrap for supply to a user who wishes to perform an assay. Such a carrier system may also be supplied in a sterile medium, which may be liquid or gas. Alternatively, as described above, the carrier system may be provided pre-loaded with an assay sample such as a frozen cell, which is usually in a state ready for use in an assay. In such a carrier system, the carriers may be appropriately marked for identification, such as for identifying individual carriers or groups of carriers.

[0105] PCT / GB2019 / 053188, a co-pending application filed by the present applicant, describes the fabrication of carriers defined by lithography for multi-channel assays. In the context of the present invention, the process described in PCT / GB2019 / 053188 needs to be modified to not use a water-soluble photoresist, but many of the features described in PCT / GB2019 / 053188 are directly applicable to the present invention. For example, SU-8 photoresist can be used. PCT / GB2019 / 053188 is hereby incorporated by reference in its entirety and reproduced below in the appendix.

[0106] Note that in the prior art, different types of magnetic particles for use in assays have been described. For example, patent publication WO2009 / 029859 describes a method for forming magnetic nanodisks with a width from 1 nm to 200 nm by electrodeposition or chemical vapor deposition. The nanodisks are formed on a layer of sodium chloride or potassium chloride, dissolved in water, or formed on a layer of copper, silver, or aluminum and dissolved in an acid / metal etching solution. The product of this method is free nanodisks in a solution provided to the user for use in a DNA array. (The solution may be the solution used to remove the nanodisks from the salt layer, or the nanodisks may be washed and redispersed in a suitable solution for use in the DNA array.) The nanodisks are formed with appropriate molecules on their surfaces and can bind to specific desired targets, i.e., specific structures on the surface of cells, when the nanodisks are in solution, and those targets can be identified by the use of a magnetic field sensor that senses the magnetic field of the nanodisks. The nanodisks are too small to carry or support cells and cannot be used in an assay on a substrate because they are too small to support cells. The nanodisks can be individually affected by an external magnetic field, but the volume of the magnetic material within such nanodisks is insufficient to orient an assay sample, such as cells in an assay, even after the nanodisks have bound to the assay sample.

[0107] Patent Publication US2013 / 0052343 describes another type of magnetic particles for targeting assay samples in solution. These particles have diameters ranging from 2 or 3 micrometers up to 100 micrometers or 500 micrometers and are formed by deposition in a pattern defined by a layer of photosensitive resin deposited on top of a layer of polymethyl methacrylate resin (PMMA). The photosensitive resin is selectively removed in the areas where the particles are to be formed, the particles are deposited, and then the PMMA is dissolved in a solvent such as acetone to release the particles. The particles are then washed and suspended in a suitable solution for use. The particles are provided to the user in such a form to enable targeting and recognition of desired molecular or cell species.

Brief Description of the Drawings

[0108] Here, specific embodiments of the present invention will be described by way of example with reference to the accompanying drawings.

Figure 1

Figure 2

Figure 3

Figure 4

[0109] Sheets 5 to 13 of the accompanying drawings reproduce the drawings of PCT / GB2019 / 053188, a co-pending application incorporated herein by reference in its entirety, and are reproduced below in the appendices.

DETAILED DESCRIPTION OF THE INVENTION

[0110] A carrier system 100 according to a first embodiment of the present invention is shown in FIG. 1. The carrier system 100 includes a substrate 10 in the form of a silicon chip. Four carriers or particles 12 are fixed to the substrate 10 by a release layer 14 containing dextran. The carrier system can be used in an assay of adherent cells. Each of the carriers 12 is suitable for receiving one adherent cell or a plurality of adherent cells. Each carrier is in the shape of a cube with a relatively large, square upper and lower surfaces, a flat or high aspect ratio. The upper surface 16 of each carrier, on the side opposite to the lower side of the carrier in contact with the release layer, is adapted to the cells to be adhered. The carriers have a lateral dimension of 100 microns. Each carrier includes a layer of magnetic material not shown in FIG. 1.

[0111] Each carrier includes a barcode 18 such as a Quick Response (QR) code or a two-dimensional data matrix code. A predetermined readable code is assigned to each carrier to identify the carrier. For example, a predetermined barcode 18 may refer to a specific adherent cell received on the carrier 12, or a specific adherent cell and a specific reagent to which the adherent cell is exposed as part of an assay. In other words, the barcode 18 enables a multi-channel assay to be performed by the carrier 12.

[0112] As shown in FIG. 1, since the release layer 14 is discontinuous, the substrate is exposed 15 between each of the carriers 12. However, during manufacture, the release layer 14 was originally formed by spin-coating a continuous layer of dextran covering the entire surface of the substrate 10, and the portions of the release layer between the carriers were removed after the carriers were formed. Thus, the release layer 14 under the carriers can be regarded as a single layer.

[0113] Figure 1 shows four carriers 12 fixed to a substrate 10, although any number of carriers 12 can be fixed to the substrate. A single carrier 12 can be fixed to the substrate.

[0114] A lithography process for manufacturing a carrier system 100 according to an embodiment of the present invention is shown in FIG. 2.

[0115] FIG. 2a shows a release layer 14 formed on a silicon substrate 10 by spin-coating an aqueous solution containing dextran 70 in an amount of 20% by weight onto the substrate. After spin-coating the release layer, the silicon substrate 10 is baked. In the case of the dextran 70 release layer, the bake is at 150° C. for 2 minutes. A photoresist layer 20 is formed on the release layer 14 by spin-coating SU-8 photoresist onto the release layer 14. The SU-8 photoresist is then baked.

[0116] In an alternative embodiment, the release layer 14 can be formed from polyvinyl alcohol instead of dextran 70 by spin-coating an aqueous solution containing polyvinyl alcohol (PVA) onto the substrate.

[0117] FIG. 2b shows a lithography patterning step performed on the SU-8 photoresist layer 20 by exposing the photoresist to ultraviolet light 24 through a patterned photomask 22 and baking the exposed resist at 95° C. for 2 minutes. This exposes specific regions of the photoresist layer 20 to ultraviolet light 24 and causes crosslinking in those regions. The remainder of the photoresist layer 20 remains soluble and can be washed away with a suitable non-aqueous solvent. This process allows discontinuities to be formed in the photoresist layer without affecting the underlying release layer and defines the base of each carrier.

[0118] Barcodes are also added during lithographic patterning of the SU-8 photoresist layer by creating an array or pattern of holes in the SU-8. These holes can be read in the transmission mode of a microscope and can function as barcodes that can be used to identify the carrier or to identify an assay sample received on that carrier.

[0119] Figure 2c shows the photoresist layer 20 after exposure to a suitable solvent. The main solvents for SU-8 photoresist are PGMEA, γ-butyrolactone or cyclopentanone. The soluble, uncrosslinked regions of the photoresist are washed away by the solvent, creating a discontinuous SU-8 photoresist layer. Since γ-butyrolactone or cyclopentanone are non-aqueous solvents, washing away the uncrosslinked regions of the photoresist layer does not dissolve the release layer 14. The release layer 14 is not soluble in non-aqueous solvents such as γ-butyrolactone or cyclopentanone. After washing, the remaining SU-8 forms the base 26 of each carrier.

[0120] Next, oxygen plasma etching is performed using the photoresist as a mask to remove the release layer 14 from the substrate 10 in the regions between the carriers. Figure 2d shows the overlapping portions of the release layer and the photoresist layer that form the base 26 of the carriers after this etching process.

[0121] Figure 2e shows the carrier system after a further layer 28 containing a magnetic material has been deposited on the photoresist base 26 of the carrier. These layers 28 include a plurality of magnetic material layers interspersed with non-magnetic material layers and are added by magnetron sputtering. To ensure that the carrier has a low stray magnetic field to avoid carrier aggregation, a layer structure comprising 11 layers is used, with the layers being, as follows, Au(20.0) / Ta(2) / Pt(4) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(1.2) / Au(20.0) / Ta(2) / Pt(4) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(5.0) (thickness in nm). The SU-8 photoresist layer of the carrier is about 1.5 to 2 microns thick and it should be noted that, advantageously, it provides mechanical stability to the carrier. Thus, the magnetic layer is mechanically supported.

[0122] Next, a gold cap is formed on the carrier using a top-down lithography process. The gold cap is deposited on the surface of the carrier that is exposed while the carrier is fixed to the substrate. The gold cap can provide biocompatibility and further, a coating or surface modification can be applied to the gold cap depending on the desired assay application of the carrier.

[0123] Figure 2f shows the carrier system after the gold cap layer has been adapted to be particularly suitable for receiving adherent cells by applying a coating 29 containing a polymer.

[0124] The polymer contains thiol groups and is applied by immersing a carrier system in a solution containing the polymer for between 20 and 120 minutes. The solution contains the polymer at a concentration between about 10 μM and 10 mM. The polymer covalently bonds via the thiol groups to the gold cap of the carrier. The solution contains a non-aqueous solvent such as ethanol. Since the release layer is not soluble in such non-aqueous solvents, the carrier remains fixed to the substrate while the carrier system is immersed in the solution containing the polymer. Figure 3a shows a schematic view of a carrier 12 comprising a coating of a charged polymer 32 containing thiol groups covalently bonded to the carrier 12.

[0125] Alternative coatings can be applied to the carrier. Instead of a charged polymer, the coating 29 can include a plurality of ligands including an antibody that specifically binds to a cell receptor such as an integrin, or an extracellular matrix protein such as collagen or Matrigel, a protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Figure 3b shows a schematic view of a carrier 12 having a coating applied thereto that includes a plurality of ligands 34.

[0126] Instead of applying a coating, the method can include modifying the physical surface of the carrier so that it is particularly suitable for receiving adherent cells, for example, by modifying the gold cap. Figure 3c shows an embodiment in which the physical structure of the carrier, and in particular the gold cap, is adapted for receiving adherent cells. In this embodiment, the surface area is increased by providing grooves 30 on the upper surface of the carrier. The grooves 30 are formed by an additional lithography step or by etching the carrier (e.g., by plasma etching) during the manufacturing process, prior to the deposition of gold. The physical structure or topography of the surface can affect the extent to which adherent cells adhere or seed on the surface. Cell adhesion can be enhanced by increasing the surface area of the surface of the carrier.

[0127] Although not shown, the final step in the method of manufacturing the carrier system is sterilization. To sterilize the carrier system, the carrier system is immersed in pure ethanol for 20 minutes. This eliminates unwanted types of living organisms or biological agents that could otherwise interfere with the assays performed using the carrier system. As a reminder, since dextran or polyvinyl alcohol is not soluble in such non-aqueous solvents, the carrier remains fixed to the substrate while the carrier system is being sterilized.

[0128] Figure 4 shows a carrier system used for the assay of adherent cells 42 and includes the process of introducing a biocompatible aqueous solution 40 and adherent cells 42 into the carrier system 100. The adherent cells 42 received by the carrier 12 are transported in the biocompatible aqueous solution to maintain viability. Once the adherent cells are received by the carrier, the cells reach the desired or natural adhesion configuration. The coating 29 on the carrier, as described above, improves the efficiency or rate at which the cells are received by the carrier, thus shortening the time required for the cells to be received on the carrier and increasing the strength of the bond or attraction between the assay sample and the carrier. Advantageously, therefore, the assay sample is securely bound to the carrier.

[0129] In practice, the carrier system 100 will be placed in a container and immersed in the biocompatible aqueous solution. In Figure 4a, this is schematically represented by a test tube 44 containing an amount of the biocompatible aqueous solution that includes drops of adherent cells 42 and biocompatible aqueous solution 40. The biocompatible aqueous solution 40 and adherent cells 42 are not drawn to scale.

[0130] When the biocompatible aqueous solution 40 contacts the dextran-containing release layer 14, the release layer 14 dissolves. This releases the carrier 12. Since dextran is biocompatible and non-toxic, dissolving the dextran release layer 14 in the aqueous solution 40 has a minimal impact on the adherent cells 42, and thus has a minimal impact on the results of the assay to be performed on the adherent cells 42. FIG. 4b shows the release layer 14 that has completely dissolved to release the carrier 12. However, in other embodiments using different materials for the release layer, the release layer 14 may not dissolve or may not completely dissolve. In such cases, it is only required that the bond between the release layer 14 and the carrier 12 be weakened so that the carrier 12 can be released.

[0131] Typically, in the presence of a biocompatible aqueous solution, the dextran release layer 14 will release the carrier in 5 seconds or less. This is significantly shorter than the typical time required for adherent cells to be received by the carrier. It may be necessary for adherent cells to take more than 3 hours to be received by the carrier 12. However, it is preferred that the adherent cells be received on the carrier while the carrier is in contact with the substrate. This ensures that the outer surface of the carrier 12, i.e., the upper surface 16, remains the main exposed surface of the carrier and prevents the adherent cells 42 from being received, for example, on the bottom surface of the carrier 12.

[0132] As shown in FIG. 4b, an external magnetic field is applied to hold the carrier in contact with the substrate even after the release layer has released the carrier. As described above, the carrier 12 includes a layer 28 containing a magnetic material. The external field applies a force to the carrier 12 that presses the carrier against the substrate and is arranged to hold the carrier in contact with the substrate 10 even after the release layer 14 has released the carrier 12. The external field is represented by the dotted arrow in FIG. 4b.

[0133] In an alternative embodiment, the release layer can be designed to match or exceed the typical time for adherent cells to be received on the carrier, taking into account the release time. In that case, as shown in Figure 4b, the step of applying an external field may not be necessary. A release layer containing polyvinyl alcohol can be biocompatible and made to have a longer release time, such as 12 hours or more. Advantageously, this can be achieved by a combination of the formulation of the PVA layer (degree of hydrolysis in the range of 85 - 99%, or the ratio of two PVAs with different degrees of hydrolysis) and typically baking at about 115 °C. If the baking step is omitted, a PVA layer with a very short release time, up to a few minutes or less, can be produced. In this way, the release time of the release layer can be designed as required for a particular assay application.

[0134] Thus, typically, a carrier system 100 including a properly fabricated polyvinyl alcohol release layer 14, when used with adherent cells that take 3 hours to be received by the carrier 12, as described above, may not require applying an external field and holding the carrier in contact with the substrate.

[0135] When the carrier is released from the substrate, the released carrier 12 can freely move in the aqueous solution 40 surrounding the carrier system. This is shown in Figure 4c. The external magnetic field can be used to manipulate, move the carrier as desired, and perform the assay, and can apply a force to the carrier. The biocompatible aqueous solution 40 also provides a suitable environment for the adherent cells 42 to maintain their viability. In particular, since adherent cells are most viable when received on a surface, it is more convenient to manipulate and transport adherent cells received on the carrier rather than trying to manipulate and transport the adherent cells themselves. Receiving cells on the carrier 12 allows each adherent cell to be transported as if it were in suspension while remaining in a biologically representative adherent state.

[0136] When a carrier having adherent cells received thereon is detached from a substrate, an assay can be performed.

[0137] In summary, the numbered clauses below describe various preferred embodiments of the present invention. 1. A carrier system for an assay comprising particles or a carrier fixed to a substrate by a release layer, wherein the particles or the carrier are suitable for receiving an assay sample, and the release layer is configured to release the particles or the carrier from the substrate in the presence of a biocompatible aqueous solution during use.

[0138] 2. The carrier system according to clause 1, wherein the release layer is configured such that the biocompatible aqueous solution maintains biocompatibility after activation of the release layer.

[0139] 3. The carrier system according to clause 1 or 2, wherein the release layer comprises a water-activatable material such as a water-soluble material.

[0140] 4. The carrier system according to any one of clauses 1 to 3, wherein the release layer is not activatable in a non-aqueous solvent such as ethanol.

[0141] 5. The carrier system according to any one of clauses 1 to 4, wherein the release layer comprises at least one of a sugar such as dextran or polyvinyl alcohol.

[0142] 6. The particles comprise a magnetic material, preferably the particles comprise a layer structure between the upper surface of the particles and the opposing bottom surface of the particles, the layer comprising one or more magnetization layers, and particularly preferably, the ratio of the lateral dimension of the one or more magnetization layers to the thickness or collective thickness of the one or more magnetization layers is greater than 500. The carrier system according to any one of clauses 1 to 5.

[0143] 7. The carrier system according to any one of clauses 1 to 6, wherein the particles are defined by lithography.

[0144] 8. The particle is the carrier system according to any one of clauses 1 to 7, including a photoresist layer such as SU-8 photoresist.

[0145] 9. The surface of the particle is adapted to receive the assay sample. For example, the surface includes a gold cap layer to which a polymer is covalently bonded by a thiol group, and is the carrier system according to any one of clauses 1 to 8.

[0146] 10. The particle includes readable information such as a readable code selected from a barcode or a two-dimensional code, and is the carrier system according to any one of clauses 1 to 9.

[0147] 11. The carrier system according to any one of clauses 1 to 10, including a plurality of particles, each of which is fixed to the substrate by the release layer.

[0148] 12. A method for manufacturing a carrier system for an assay, comprising: providing a substrate; forming a release layer on the substrate; depositing a particle or carrier for receiving an assay sample on the release layer such that the particle or carrier is fixed to the substrate; and the release layer is configured to release the particle from the substrate in the presence of a biocompatible aqueous solution during use. Method.

[0149] 13. The method according to clause 12, wherein the step of forming the release layer on the substrate includes spin-coating the release layer.

[0150] 14. The method according to clause 12 or 13, wherein the step of depositing the particle on the release layer includes fabricating the particle on the release layer, for example, by a lithography process.

[0151] 15. The method according to any one of clauses 12 to 14, wherein the release layer is adapted to release the particles from the substrate in the presence of the biocompatible aqueous solution within a time period of from 1 hour to 72 hours during use.

[0152] 16. The method according to any one of clauses 12 to 15, further comprising the step of adapting the surface of the particles such that the surface is suitable for receiving the assay sample.

[0153] 17. The method according to any one of clauses 12 to 16, further comprising the step of sterilizing the carrier system by immersing the carrier system in ethanol after the step of depositing the particles on the release layer.

[0154] 18. The method according to any one of clauses 12 to 17, wherein the step of depositing the particles comprises forming a magnetic structure.

[0155] 19. A method of performing an assay using the carrier system according to any one of clauses 1 to 11, the method comprising introducing a biocompatible aqueous solution into the carrier system to release the particles or the carrier.

[0156] 20. The method according to clause 19, further comprising introducing the sample for the assay into the particles such that the sample is received by the particles.

[0157] 21. The method according to clause 20, wherein the sample is received by the particles while the particles are in contact with the substrate.

[0158] 22. The particles comprise a magnetic material, and the method further comprises applying a magnetic field to the particles, the magnetic field acting to hold the particles in contact with the substrate even after the release layer has released the particles from the substrate.

[0159] 23. The method according to clause 22, wherein the magnetic field is applied and holds the particles in contact with the substrate for at least 5 seconds, or at least 1 minute, or at least 5 minutes, or at least 30 minutes.

[0160] 24. A method of using the carrier system according to any one of clauses 1 to 11, comprising introducing the sample for assay into the particles or carrier such that the sample is received by the particles while the particles are in contact with the substrate, and preferably storing the sample received on the particles or carrier by freezing the sample received on the particles.

[0161] 25. The method according to clause 24, further comprising the step of detaching the particles from the substrate before storing the sample received on the particles.

[0162] [Appendix] For reference, the content of International Patent Application PCT / GB2019 / 053188, a co-pending application filed by the applicant, is reproduced below. This includes a detailed description of the production of magnetic carriers or particles similar to those in an embodiment of the present invention, with the difference that in the present invention, the carrier is produced on or adheres to the release layer. When the carrier is produced on the release layer, a lithography process that does not activate the release layer prematurely needs to be used instead of that described in the appendix. One such option is to replace the photoresist described in the appendix with a photoresist that can be washed away without using an aqueous solvent. The figures of PCT / GB2019 / 053188 are described on sheets 5 / 13 to 13 / 13 of the drawings.

[0163] (PCT / GB2019 / 053188: Magnetic Carrier and Method) The present invention relates to a magnetic carrier, a method of manufacturing a magnetic carrier, and a method of using a magnetic carrier.

[0164] (PCT / GB2019 / 053188: Background Art) Techniques for using an applied magnetic field to exert a mechanical force on individual magnetic carriers are utilized in various biotechnology applications.

[0165] Currently, one important commercial use of magnetic nano- and micro-carriers is for bioassays for separating and identifying biomolecules. Superparamagnetic iron oxide nano-carriers (SPIONs) have conventionally been used for this commercial range of applications because they provide the property of moving towards an external magnetic field source. This enables the carriers to be induced towards the desired position for reading assay information by the application of an external magnetic field. These carriers are nano-carriers (5 - 20 nm in diameter) made by colloidal chemical methods. The size of these carriers is limited by the fact that if the carriers become larger than approximately 20 nm, the carriers become ferromagnetic and, disadvantageously, the influence of the stray magnetic field of one carrier on other carriers results in magnetic aggregation of the carriers, preventing their use in bioassays.

[0166] Engineers developing magnetic carriers need to optimize the magnetic properties of the carriers for each application. Conventionally, it has been common knowledge that a highly desirable property of magnetic nano-carriers for all these various applications, including bioassay applications, is a zero net magnetization remanence state in order to avoid aggregation of the carriers.

[0167] A net-zero remanent magnetization state means that in the absence of a magnetic field, the magnetic carriers have no net magnetic moment and no external stray magnetic field. In use, typically, the magnetic carriers are suspended in a liquid or liquid medium and can move freely within that medium. In the case of carriers with non-zero remanent magnetic moments, the stray magnetic fields of the carriers can interact and cause the carriers to aggregate or clump together. Since the purpose of using magnetic carriers in biotechnology applications is that the movement of the carriers suspended in a liquid or liquid medium can be induced or directed by applying an external magnetic field, this is not desirable. If the magnetic carriers aggregate, this may not be achieved.

[0168] Furthermore, it is understood by those skilled in the art that in order to ensure that small magnetic fields from the environment cannot cause aggregation by inducing magnetic moments in the magnetic carriers, the carriers having a zero-net remanent magnetization state should also have a low magnetic susceptibility in small magnetic fields.

[0169] Furthermore, if carriers with high magnetic susceptibility are used, after an applied magnetic field is applied to direct or move the carriers in a desired manner and then the applied magnetic field is removed, the carriers aggregated during the application of the magnetic field will remain aggregated. This is also understood by those skilled in the art to be something to be avoided in magnetic carriers for biotechnology applications.

[0170] Therefore, prior research in this technical field to produce non-aggregating magnetic nanocarriers has focused entirely on systems having a zero-net remanent magnetization state and preferably having a low magnetic susceptibility. This includes various systems such as superparamagnetic nanocarriers, magnetic vortex micro-nanocarriers, and micro-nanocarriers that utilize antiferromagnetic coupling to generate opposite magnetization alignments between adjacent magnetic layers of the carriers.

[0171] An important biotechnology application is to perform multiplexed immunoassays on biological samples. Accurate quantification of proteins in biological samples is highly important for both research and clinical diagnostic applications. Multiplexed immunoassays simultaneously quantify multiple different proteins in a given sample. Analyzing the protein fingerprint of a sample in this way has the potential to accelerate research and enable improved diagnosis. In response to this market need, multiplex assay systems such as Luminex(RTM), Firefly(RTM), and Fireplex(RTM) have been developed. These systems use individual carrier sets where each carrier is coated with a capture antibody suitable for one specific analyte. Multiple sets of carriers specific for the analytes can then be combined and multiple targets simultaneously detected and quantified through the use of detection antibodies marked with fluorescent labels. The Luminex(RTM) system is based on polystyrene or paramagnetic microspheres, or beads, internally stained with different intensities of red and infrared fluorophores, allowing one set of beads to be distinguished from another. The Firefly(RTM) and Fireplex(RTM) systems also use fluorophores and can distinguish one carrier set from another, but in this case the carriers are in the form of rods encoded by applying different fluorophores to each end. Again, the measurement of the fluorophores is aimed at distinguishing one rod from another. However, in practice, these systems suffer from limited multiplexing (limited number of different proteins that can be distinguished) due to their limited ability to reliably distinguish between multiplexed channels in assay results.

[0172] (PCT / GB2019 / 053188: Summary of the Invention) The present invention provides a magnetic carrier, a plurality of magnetic carriers for performing an assay, and a method for performing an assay using the magnetic carriers, as defined in the appended independent claims, which are hereby incorporated by reference. Preferred or advantageous features of the invention are set out in the dependent subclaims.

[0173] Accordingly, in a first aspect, the present invention may provide a magnetic carrier, a layer structure between an upper surface of the carrier and an opposing bottom surface of the carrier, and one or more layers including one or more magnetized layers. The ratio of the lateral dimension of the one or more magnetized layers to the thickness of the one or more magnetized layers, or the collective or effective thickness, is greater than 500. In other words, the aspect ratio of the cross-section of the one or more magnetized layers may be greater than 500. In a preferred embodiment, the ratio may be even higher, for example, greater than 800, or greater than 1000 or 1500 or 2000.

[0174] The carrier may further include a non-magnetic layer, and advantageously, the non-magnetic layer may provide mechanical support to the magnetic layer and may determine physical properties of the carrier such as its mechanical properties and its density.

[0175] The carrier may include one magnetized layer or may include two or more such layers. When the carrier includes two or more magnetized layers, the layers may be adjacent to each other, or in contact with each other, or may be separated from each other with a non-magnetic material therebetween. The collective thickness, or total thickness, of the remanent magnetization layers in a carrier having two or more magnetized layers may be the sum of the thicknesses of those magnetic layers and may optionally not include any non-magnetic layers therebetween. In some embodiments, the layer structure may include a number of magnetized layers and / or a number of layers of non-magnetic material.

[0176] The magnetized layer, or magnetic layer, may include any suitable one or more magnetic materials, such as, for example, ferromagnetic materials, elements or alloys, or composites of superparamagnetic nanocarriers.

[0177] Preferably, the carrier has a substantially flat shape, including one or more substantially flat layers of magnetic and / or non-magnetic materials stacked on top of each other. Preferably, the layers are substantially the same shape and size as each other, and each has the same lateral shape and size as the carrier itself. However, as will be further described below, the shape and structure of the carrier may be different from this.

[0178] The carrier may have zero or non-zero remanent magnetization. However, the inventors have found that even when the carrier has non-zero remanent magnetization, the shape and structure of the carrier embodying the present invention exhibit an unexpectedly low stray magnetic field at the surface of the carrier, and advantageously, a plurality of carriers suspended in a fluid or liquid medium may not aggregate or form clumps. Surprisingly, the inventors have found that such cases exist regardless of whether the carrier has complete remanent magnetization or not.

[0179] <� In a preferred embodiment of the present invention, when an external field is applied, the carrier has a magnetic moment sufficient for the external field to apply a desired force to the carrier. The desired force may depend on the use of the carrier (such as in a bioassay having carriers suspended in a liquid medium). In a practical embodiment, typically, the applied external field is less than 2T, or less than 1T or 0.5T, and may typically be greater than 0.05T, or 0.1T or 0.25T.

[0180] For example, in a bio or chemical assay, it may be desirable to use an external field to direct the carrier within a liquid medium. The magnetic moment may be due to the magnetization of the carrier itself, or may be induced in the carrier by an external field. However, it is important that the carrier contains sufficient magnetic material to be able to generate the desired force.

[0181] The magnetic moment that can be generated by an external field applied to the carrier may depend on the total volume V of the magnetic material in the carrier multiplied by the magnetization Ms of that material. Therefore, preferably, the value of V.Ms of the carrier embodying the present invention is 10 -18 J / T or 5x10 -18 J / T or 10 -17 J / T or greater than a predetermined value such as

[0182] The inventors have found that the physical distribution of the magnetic material within the carrier determines the stray magnetic field in the vicinity of the carrier and can therefore determine the tendency of the carriers to interact with each other and / or aggregate. The inventors have found that distributing the magnetic material in the form of one or more layers (preferably parallel layers) having a cross-section with a high aspect ratio AR can advantageously generate a low stray magnetic field. Advantageously, this preferred carrier geometry can provide carriers having a low stray magnetic field and little or no tendency to aggregate.

[0183] To more quantitatively evaluate the geometry of this carrier, the inventors have, in a preferred carrier, preferably, the parameter AR / Ms of one or more magnetic layers 2 (AR is a dimensionless ratio and Ms is the magnetization of the magnetic material in one or more magnetic layers measured in A / m (1000 A / m corresponds to 1 emu / cm 3 )) is proposed to be greater than 8x10 -10 m -2 / A -2 or 1.2x10 -9 m -2 / A -2 or 8x10 -9 m -2 / A -2

[0184] Alternatively or additionally, the inventors have preferably that the parameter AR / Ms is 1x10 -3 m -1 / A -1 or 3x10 -3 m -1 / A -1 ​or 5x10 -3 m -1 / A -1 was determined to be greater than.

[0185] These limits correspond to a stray magnetic field of less than about 2500 A / m (30 Oe) at 10 times the layer thickness, either above or below the layer. Depending on the use and environment of the carrier, advantageously, this level of stray magnetic field can prevent aggregation.

[0186] When assessing the AR value of a carrier structure, AR can be the value obtained by dividing the lateral dimension of the cross-section of the structure by the thickness of the structure. In the case of a layer of magnetic material, the lateral dimension may be the minimum lateral dimension of the layer, or, if the shape of the layer is more complex, it may preferably be regarded as the average lateral dimension of the layer. If the thickness of the layer is constant, that thickness can be used in the calculation of AR. If the thickness of the layer varies, the average thickness can be used.

[0187] For example, when the magnetic material extends across the entire lateral dimension of the carrier, particularly when the thickness of the carrier is sufficiently similar to the thickness of one or more magnetized layers, such as less than 10 times or 5 times, it may be appropriate to use the equivalent lateral and thickness dimensions of the carrier itself to calculate AR. In this approach for evaluating AR / Ms, similar to using the AR value of the carrier, the Ms value may be modified by calculating a diluted Ms value by multiplying the Ms of the magnetized material in the carrier by the volume ratio of the non-magnetic material to the magnetized material in the carrier.

[0188] The carrier may include a plurality of magnetized layers, for example, in the form of a stack of magnetized layers separated by layers of non-magnetized material. In such a case, if the magnetized layers are separated from each other at a sufficiently short distance, such as less than 5, 10, or 20 times the thickness of the thinnest layer or the average layer thickness, AR may be evaluated using either the collective thickness of the magnetized layers including the thickness of any intervening non-magnetic layers or the distance between the outermost magnetized layers of the stack.

[0189] If the carrier includes a plurality of layers that extend over a sufficient proportion of the carrier thickness, the AR may be evaluated using the carrier thickness.

[0190] When the carrier includes a plurality of layers, an alternative approach to evaluating the thickness for calculating the AR may be to calculate the diluted thickness of the magnetized material. For example, if the collective thickness Tm of two or more parallel layers of magnetized material is separated by the collective thickness Tnm of layers of non-magnetic material, the diluted thickness of the magnetized material is Tm / (Tm + Tnm).

[0191] By measurement, it may also be possible to evaluate the AR and Ms for the calculation of (AR / M S 2 or AR / M S of the entire carrier). If the magnetic moments of a predetermined number of carriers of unknown structure but known dimensions are measured (e.g., using a vibrating sample magnetometer), the effective Ms may be found using the total volume of the carriers and the total moment per carrier. The lateral dimensions and thickness of the carriers or metal layers may be directly measured, for example, using microscopy and / or electron microscopy techniques, and the AR may be evaluated.

[0192] Preferably, the carrier embodying the present invention has a planar shape, and both its length and width are greater than its thickness. Advantageously, the length and width of the carrier, or two lateral dimensions of the carrier measured perpendicular to each other, are similar to each other, or differ from each other by less than about 10%, 30%, 50% or 70%. Typically, the carrier may be in the form of a circular or elliptical or polygonal disk, or a generally flat cube having a square or rectangular perimeter.

[0193] Preferably, the magnetic material within the carrier is in the form of one or more layers within the carrier and extends substantially across the entire lateral dimension of the carrier. The aspect ratio of the one or more magnetic layers can be assessed with reference to the minimum lateral dimension, or the average lateral dimension, of the one or more layers, and can be the same as the lateral dimension of the carrier or smaller than the lateral dimension of the carrier. When there is one magnetized layer, the aspect ratio AR can be the value obtained by dividing the minimum, or average, lateral dimension of the magnetic layer by its thickness (or average thickness if the thickness varies). When there are multiple magnetized layers, the aspect ratio AR can be evaluated as the value obtained by dividing the lateral dimension, or the average lateral dimension if different layers have different lateral dimensions, by the collective thickness of the layers.

[0194] In a preferred embodiment, the top and bottom surfaces of the carrier may be separated by the thickness of the carrier between 5 nm, or 10 nm or 50 nm or 100 nm and 100 μm or 50 μm or 5 μm or 1 μm or 500 nm. The minimum lateral dimension of the carrier may be greater than 1 μm, preferably greater than 5 μm or 10 μm, and the maximum lateral dimension may be less than 500 μm or 200 μm or 100 μm or 50 μm. The ratio of the minimum lateral dimension of the carrier to the thickness of the carrier may be greater than 10 or 20 or 50 and / or less than 2000 or 1000 or 500. Thus, in such preferred embodiments, the carrier may have a rather flat, high aspect ratio shape, while other embodiments envision carriers with lower aspect ratio shapes, or even spherical or cubic carriers. Such low aspect ratio carriers may include one or more magnetized layers having a higher aspect ratio, as discussed above and herein.

[0195] In embodiments including two or more magnetized layers, preferably, those layers are substantially parallel to each other. In embodiments including two or more magnetized layers, preferably, those layers have similar shapes and / or areas to each other, and for convenience, they may overlap each other, and optionally, they may completely overlap each other.

[0196] Advantageously, the opposing upper and lower surfaces of the carrier are flat, but optionally, one or both surfaces may be curved or not flat without affecting the desired properties of the carrier having a sufficiently small levitation magnetic field to avoid aggregation. Thus, the carrier itself may be flat or curved. However, in each case, advantageously, the opposing upper and lower surfaces are such that individual carriers or groups of carriers (when the carriers within the group of carriers are similarly marked) can be identified by reading information, so that features such as readable information in the form of a barcode or two-dimensional code are applied to the upper and / or lower surfaces. Such information may be applied to the upper and / or lower surfaces, or under the upper and / or lower surfaces, for example, under one or more surface layers that are sufficiently transparent to allow the code or information to be read through the one or more surface layers. Further, as described herein, advantageously, the upper or lower surface may form a suitable substrate for other functional applications to the carrier, such as biocompatibility or chemical functionality for biotechnology or chemical applications.

[0197] Thus, in a preferred embodiment, the shape of the carrier may be a thin (small thickness) laterally extending form, such as a cube or disk with a high aspect ratio. (The aspect ratio means the ratio of the minimum lateral dimension, or average lateral dimension, to the thickness). Alternatively, the magnetic carrier may be cylindrical in shape, the thickness of the carrier being in the axial direction of the cylinder, and preferably, the peripheral shape of the cylinder may be described as being selected such that it typically has one or more edges that are convex or linear and advantageously have no recessed corners. Preferred peripheral shapes are rectangular or square or circular.

[0198] As described above, the carrier has a high aspect ratio or a cylindrical shape, and preferably, the carrier is flat and has a flat upper surface and a bottom surface. While embodiments of the present invention achieve the object of providing a non-aggregating magnetic carrier, it is contemplated that the carrier may include a curved or non-flat carrier, or a carrier having a curved or non-flat upper surface and a lower surface.

[0199] Preferably, the minimum and maximum lateral dimensions of the magnetic carrier differ by less than 90% or less than 70%. In a preferred embodiment, the minimum lateral dimension of the carrier is greater than 5 μm, preferably greater than 10 μm, and / or the maximum lateral dimension of the carrier is less than 500 μm, preferably less than 200 μm, 100 μm or 75 μm. These dimensions may be selected by those skilled in the art according to requirements such as the application for which the carrier is used and the desired mechanical strength of the carrier.

[0200] Advantageously, the layer structure of the magnetic carrier includes a magnetization layer and a non-magnetic layer. The non-magnetic layer may impart mechanical strength to the carrier and may provide a suitable substrate for the magnetization layer. Thus, advantageously, the non-magnetic layer may include materials selected from AI, Ta, Pt, Pd, Ru, Au, Cu, W, MgO, Cr, Ti, Si, Ir, SiO2, SiO, Sn, Ag, polymers, plastics, alloys of these materials, and composites or mixtures containing these materials.

[0201] The carrier may include two or more layers of non-magnetic material and may similarly be selected from this group.

[0202] The magnetization layer, or ferromagnetic layer, may be formed from any suitable material and, in a preferred embodiment, may include materials selected from metals or metal alloys such as, for example, Fe, Co, Ni, CoFe, CoFeB, FePt, CoNi, and NiFe.

[0203] For example, the magnetization layer may include a magnetic multilayer stack of alternating layers of a magnetic material and a noble metal (such as Pt / CoFeB), and this pair is known to provide perpendicular magnetic anisotropy.

[0204] Preferably, the magnetized layer is an out-of-plane magnetization layer, but it may also be a different magnetization layer such as an in-plane magnetization layer.

[0205] [[ID=A]] To achieve a rapid response to an external magnetic field, a high saturation magnetic moment is desirable for the magnetic carriers. The magnetic material is selected to achieve this.

[0206] The layer structure of the magnetic carrier may include two or more layers of a non-magnetic material and / or two or more layers of a magnetized material. In a preferred embodiment, the carrier may include a magnetization layer disposed between two layers of a non-magnetic material.

[0207] The carrier may include two or more magnetization layers arranged in combination so as to have zero remanent magnetization when no applied magnetic field is present. Such a carrier may have a magnetic susceptibility such that the application of an external field induces a magnetic moment in the carrier. Thus, the external field may be applied, for example, via a liquid medium, to move or guide the carrier. However, advantageously, the shape of the carrier embodying the present invention may be such that even when a magnetic moment is induced, the stray magnetic field around the carrier is advantageously too low to cause aggregation of the carriers, so it may not be important whether the carrier has a high or low magnetic susceptibility.

[0208] Advantageously, in such a carrier, the magnetization layer is separated from the top surface of the carrier by more than 25% of the thickness of the carrier and may be separated from the bottom surface of the carrier by more than 25% of the thickness of the carrier. Advantageously, this structure may further reduce the stray magnetic field at the opposing top and bottom surfaces of the carrier.

[0209] Preferably, the magnetization layer may have a thickness or an average thickness greater than 0.1 nm, or 0.4 nm, 1.0 nm or 1.5 nm. Preferably, the thickness of the magnetization layer may be less than 25%, particularly preferably less than 15% or 10% of the total thickness of the carrier. If the mechanical strength of the carrier is sufficient for the desired application, the carrier may comprise only the magnetization layer.

[0210] For example, the magnetization layer may be a thin film multilayer.

[0211] At a short distance or within a short distance from the upper or bottom surface of the carrier, the net magnetic field (floating magnetic field) averaged over the lateral surface is preferably less than 2500 A / m (30 Oe), particularly preferably less than 800 A / m (10 Oe) or less than 400 A / m (5 Oe). This magnetic field may be measured at the surface, for example, by using a magnetic atomic force microscope, or at a short distance such as 10 nm, 50 nm, 100 nm from the surface. The experiments of the present inventors have shown that these external magnetic fields, or floating magnetic fields, are small enough to avoid aggregation of the magnetic carriers.

[0212] For convenience, the magnetic carriers embodying the present invention may be manufactured or fabricated by a lithography process.

[0213] As described above, advantageously, the second aspect of the present invention may provide a magnetic carrier having dimensions, preferably, the upper surface of the carrier and the opposing bottom surface of the carrier are separated by the thickness of the carrier between 5 nm and 200 μm, the minimum lateral dimension of the carrier is greater than 1 μm, the ratio of the minimum lateral dimension to the thickness is greater than 10, the carrier includes a layer structure by its thickness, and the layer includes one or more magnetization remnants, or magnetization layers, and one or more layers of non-magnetic materials. For convenience, such a carrier may be fabricated by a lithography process and may include one or more of the features of the first aspect of the present invention described herein.

[0214] In a further aspect of the present invention, the upper or lower surface of the carrier may carry readable information, such as a readable code. For example, this may be a barcode or a two-dimensional code. This may enable the carrier to be remotely identified, for example, by reading the information using a camera and appropriate software.

[0215] In a preferred embodiment, the magnetic properties of the carrier enable an appropriate external magnetic field to be applied to guide, move, or drive the carrier through a liquid medium to a predetermined position for reading the code or information. For example, a carrier having a high aspect ratio shape with a large upper or lower surface on which the code or information is carried may be oriented to be in contact with a substrate or other support surface for easy reading of the code or information.

[0216] In a further aspect of the present invention, the upper and / or lower surface of the carrier may be functionalized, for example, bio-functionalized or chemically functionalized. Advantageously, this may be combined with applying readable information to the carrier. For example, the upper or lower surface of the carrier may carry a readable code, and the same or opposite surface may be functionalized. Further, in a preferred embodiment, a plurality of carriers are provided, and each carrier may carry readable information corresponding to the functionalization of that carrier.

[0217] Such carriers may enable the performance of an assay, such as a bioassay, by providing the carrier to a liquid or fluid assay sample and interacting the functionality of the carrier with the assay sample, for example, a biomolecule or other component of the assay sample. A magnetic field may be applied to guide the carrier to a reading position, and the assay result may be obtained by reading the readable code and measuring the interaction between the functionality of the carrier and the assay sample.

[0218] A multi-channel assay can be performed by providing a plurality of carriers, each carrier carrying readable information corresponding to a different functionality of the carrier. The plurality of carriers may include groups of carriers, and the carriers of each group may carry similar readable information and may be similarly functionalized. The plurality of carriers can be contacted with a liquid or fluid assay sample, and the functionality of the carriers can be made to interact with the assay sample. A magnetic field is applied to direct the carriers to a reading position, and assay results are obtained by reading the readable information of two or more carriers and measuring the respective interactions of the corresponding functionality of each carrier with the assay sample.

[0219] Advantageously, the identification of carriers using readable information in such a manner can provide a multiplex platform in which carriers can be accurately distinguished from one another. For example, the use of barcodes, or two-dimensional codes, can provide a significantly more robust process for distinguishing different carriers with minimal crosstalk between multiplex channels than existing multiplex assay platforms. Further, the use of readable information in such a manner can enable the use of a much greater number of multiplex channels than is currently possible. For example, barcoding or two-dimensional codes may enable 1000-plex, or 10,000-plex, or more as required.

[0220] Thus, in a preferred embodiment, advantageously, the present invention can relate to carriers defined by lithography and magnetized vertically (or out-of-plane) in the form of ferromagnetic microdisks (microcarriers, nanocarriers, microcarriers, etc.) for use in biotechnology applications. For example, these carriers can be ferromagnetic microcarriers or microdisks produced by photolithography and physical vapor deposition of magnetized thin film multilayers (in each lateral dimension, or in two orthogonal lateral dimensions, between 1 - 500 μm, or between 1 - 200 μm, or preferably between 5 - 100 μm, between 10 nm - 200 μm, or preferably, with a thickness between 20 nm and 10 μm). For example, the carrier can be circular or square with a diameter or side length of 40 μm and a thickness of 100 nm. Alternatively, the carrier can have a diameter or side length of 100 μm and a thickness of 1 μm. The resulting high planar aspect ratio ultra-thin disk, or microdisk (which can be called a magnetic carrier (MC) because of its ability to carry functionalization such as a biofunctional antibody for diagnostic tools) is a ferromagnetic material having a high magnetic moment. The MC can be defined by lithography. The aspect ratio of each magnetic layer (typically, the total thickness of the magnetic layer of 1 nm, or 5 nm and a lateral size of several tens of μm), and the magnetization direction perpendicular to the plane of the MC results in a negligible stray magnetic field from each carrier, so that it does not aggregate when suspended in a fluid.

[0221] In a preferred form, the MC can be characterized by a magnetization direction parallel to the normal of the microdisk surface, as well as magnetization reversal by coercive force and high magnetic anisotropy. All of these properties can enable a high degree of control over the magnetic response, and thus the mechanical behavior, in a fluid under the influence of an external magnetic field.

[0222] Preferably, the physical vapor deposition process used to create the carrier, i.e., the MC, enables sub-nanometer control in the deposition of the magnetic thin film forming the MC, and thus provides extremely high precision in the engineering of the magnetic properties of the MC. Advantageously, this may enable them to be tailored for different applications. Further, a barcode (or other readable information) may be added to the surface of the MC by lithography, and the surface material may be selected for optimal functionalization with the molecule of interest.

[0223] (PCT / GB2019 / 053188: Specific Embodiments and Best Modes of the Invention) Here, specific embodiments of the present invention will be described by way of example with reference to the accompanying drawings.

[0224] Figure 1 shows the steps in two processes, Process A and Process B, for the fabrication of a magnetic carrier according to the first and second embodiments of the present invention.

[0225] Figure 2 is a 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) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(5.0) used in the carrier of the embodiment.

[0226] Figures 3(a) and 3(b) show how the readable code and the magnetic state in the carrier are linked according to the embodiment, and the readable code can always be aligned with an external detector, such as a camera or a barcode reader, by an applied magnetic field, and show an image of the readable code of the carrier imaged by the detector.

[0227] Figure 4 shows the floating magnetic field strength as a function of the distance from the surface of the fabricated carrier according to the embodiment.

[0228] FIG. 5(a) and FIG. 5(b) show functionalized carriers suitable for bioassays and the use of carriers for implementing a rational multiplex assay, according to a further embodiment of the present invention.

[0229] A specific embodiment of the present invention involves the fabrication of high magnetic moment microcarriers made from ultrathin vertically magnetized CoFeB / Pt layers. The high aspect ratio of the shape of these carriers results in a very low stray magnetic field from each carrier, so that the magnetic nanocarriers do not exhibit (and thus do not aggregate) interactions between carriers. When an external magnetic field is applied, the carriers transition to magnetic saturation, have sharp switching due to the coercive force, and remain completely residual. Individual barcodes are added to the carriers using a simple and robust lithography process and can be optically read. As described below, it has been demonstrated that robust multiplex assays using magnetic carriers, such as cytokine assays, highlight their potential in assay applications.

[0230] In this embodiment, advantageously, the magnetic carriers fabricated by lithography can achieve a high magnetic moment, no interaction between carriers, a large surface area for functionalization, and specific barcoding of robust carriers. These carriers can be referred to as magnetic carriers (MC) from the perspective of their ability to carry both functionalization and readable information. Advantageously, the large surface area of the carriers can provide a larger area for functionalization than conventional assay carriers.

[0231] Magnetic nanocarriers defined by lithography are known in the prior art, for example, by 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, by H. Joisten et al., “Self-polarization phenomenon and control of dispersion of synthetic antiferromagnetic nanocarriers for biological applications,” Appl. Phys. Lett., vol. 97, no. 25, p. 253112, 2010, and by S. Leulmi et al., “Comparison of dispersion and actuation properties of vortex and synthetic antiferromagnetic carriers for biotechnological applications,” Appl. Phys. Lett., vol. 103, no. 13, p. 132412, 2013. However, in stark contrast to these lithography-defined carriers and other magnetic nanocarriers in general, the MCs used herein do not require engineering of a net-zero residual magnetization state to prevent carrier aggregation. Optionally, the MCs used herein may have a net-zero residual magnetization (and susceptibility to the generation of a magnetic moment in an external field), but contrary to the conventional expectations of those skilled in the art, they do not require a net-zero residual to avoid aggregation. Regardless of whether the residual magnetization is zero in the absence of an external field, due to the shape of the magnetized material in the carrier and / or the shape of the carrier, the stray field of the carrier is low enough to avoid aggregation.

[0232] The MC in this embodiment is a cube with a very high aspect ratio, having a planar length and width of 40 microns and a thickness of approximately 150 nanometers.

[0233] Two lithography processes (A and B) according to two embodiments of the present invention for the fabrication of magnetic carriers, i.e., MCs, are shown in FIG. 1.

[0234] Process A is shown from A1 in FIG. 1 to A11 in FIG. 1. In A1 of FIG. 1, a 50 nm Al sacrificial layer 2 is grown on the Si substrate 4 by magnetron sputtering. Next, the base 6 of the carrier thin film stack is grown on this sacrificial layer, also by magnetron sputtering.

[0235] This base is composed of the following 11 layers (thickness 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) / CoFeB(0.6) / Pt(5.0).

[0236] In A2 of FIG. 1, a photoresist 8 is spin-coated on the MC base 6. Then, as in A3 of FIG. 1, the photoresist is exposed into a lithography pattern using a photomask 10 that defines the barcode (or readable code) of the carrier. This standard photolithography process creates a plurality of holes 12 in the photoresist as shown in A4 of FIG. 1, and a barcode contrast material 14 such as 15 nm Ta is deposited on the carrier base using magnetron sputtering as shown in A5 of FIG. 1. The shape and pattern of the holes define the barcode 16.

[0237] Next, the photoresist is removed in a solvent such as acetone, and as shown in A6 of FIG. 1, a new layer of photoresist 18 is spin-coated over the carrier base 6 and barcode 16. This is exposed to a second lithographic patterning process using mask 20 to define the shape of the carrier. In this step shown in A7 of FIG. 1, a plurality of holes 22 that define the shape of the carrier are aligned such that the barcode is arranged at the center of the holes.

[0238] In A9 of FIG. 1, a carrier (MC) cap 24 and an ion beam milling hard mask 26 are added by magnetron sputtering and are composed of 30 - 40 nm of Au and 200 nm of AI respectively. The thickness of the gold is selected to ensure a full Au coating of the carrier (both on the top and bottom surfaces) for biocompatibility and to provide a surface for biofunctionalization. However, the thickness of the Au is thin enough to allow the barcode to be read through the Au layer.

[0239] Next, in A9 of FIG. 1, the photoresist 18 is removed in a solvent such as acetone, and then the entire sample is subjected to ion beam milling 28, which is a standard subtractive patterning process. The thin films not protected by the ion beam milling hard mask are removed by milling. Thus, milling removes all thin films that form the base of the carrier thin film stack that are not within the defined carrier shape. The milling process stops when the sacrificial layer is reached. Any remaining AI hard mask 26 may be removed by dissolving it by immersion in a 3 - 5% tetramethylammonium hydroxide solution, or an equivalent AI solution etchant, for 10 - 30 minutes.

[0240] Thus, photolithographic patterning determines the planar shape of the carrier, and physical vapor deposition processes determine its thickness and composition.

[0241] At this stage, the carrier 30 with the barcode, the MC, is fully defined and is on top of the sacrificial layer. Then, as shown in A10 of FIG. 1, a magnetic field 32 greater than the coercive force field of the magnetic thin film of the carrier is applied to ensure that all carriers are magnetized out of the plane in an "up" state perpendicular to the top and bottom surfaces of the carrier. Alternatively, all carriers may be magnetized in a "down" state. This links the magnetization of the carrier to the physical structure of the carrier in the vertical direction and enables barcode alignment in any downstream process such as redeposition or analysis in solution as shown in FIG. 3.

[0242] Finally, as shown in A11 of FIG. 1, the AI sacrificial layer under the carrier is dissolved in a suitable solvent to release the carrier 30 from the substrate and exfoliate them into the solution of the liquid medium.

[0243] Process B is shown from B1 to B11 in FIG. 1. In B1 of FIG. 1, the photoresist layer 50 is spin-coated on the Si substrate 4. Then, in B2 of FIG. 1, it is exposed using a photomask 52 to create a plurality of islands or pillars 54 of the photoresist. In B3 of FIG. 1, a series of material layers 56 are deposited using magnetron sputtering to form the base 58 of the thin film structure of the magnetic carrier layer. The shape of the island or pillar defines the shape of the carrier. Thus, B3 of FIG. 1 shows the structure after the deposition of the first layer of the carrier. These are shown in order from the bottom and are composed of the following thin film layers (thickness 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(5.0).

[0244] Next, the barcodes defined by lithography are added to the carriers. As shown at B4 in FIG. 1, the photoresist 60 of the second layer is applied and exposed using a photomask 62 patterned with the desired barcodes of each carrier, as shown at B5 in FIG. 1. In B6 and B7 of FIG. 1, the photoresist is developed and then flood-exposed 64 so that it can be removed in a downstream developing apparatus. The bottom layer of each photoresist island or pillar 54 is shielded from this exposure step by the presence of the carrier above the island.

[0245] At B8 in FIG. 1, a barcode contrast material 66 such as 15 nm of Ta is grown on the carrier. Then, at B9 in FIG. 1, the top layer of the photoresist is completely removed using a developer, and a carrier cap 68 composed of 30 - 40 nm of Au is deposited. The bottom layer of the resist remains as it is. The thickness of the gold is selected to ensure a full Au coating of the MC (both on the top and bottom surfaces) for biocompatibility and to provide a surface for biofunctionalization. However, the thickness of the Au is thin enough to allow the barcode to be read through the Au layer.

[0246] Thus, photolithographic patterning determines the planar shape of the carrier, and the physical vapor deposition process determines its thickness and composition.

[0247] At this stage, the carrier, MC, 70 with the barcode is fully defined and is on top of the photoresist islands. Then, as shown at B10 in FIG. 1, a magnetic field 72 greater than the coercive field of the magnetic thin film of the carrier is applied to ensure that all carriers are magnetized out-of-plane in an "up" state perpendicular to the top and bottom surfaces of the carrier. Alternatively, all carriers may be magnetized in a "down" state. This links the magnetization of the carrier to the physical structure of the carrier in the vertical direction and enables barcode alignment in any downstream process such as redeposition or analysis in solution, as shown in FIG. 3.

[0248] Therefore, the thin film structure of the MC described in this embodiment in processes A and B is defined based on 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) (thickness in nm). A 15 nm Ta barcode is on top of this layer, which is then covered with 30 - 40 nm of Au. The thinner Au on the top surface enables imaging the barcode through the Au, and thus, in the embodiments described herein, the barcode is only visible through the top surface of the carrier. Therefore, at this stage, linking the magnetization of the carrier to the physical structure of the carrier is necessary to enable control and orientation of the barcode surface of the carrier in solution.

[0249] Finally, as shown in B11 of FIG. 1, the photoresist 74 under the carrier is dissolved in a suitable solvent to release the carrier 70 from the substrate and peel them into a solution in a liquid medium.

[0250] FIG. 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) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(1.2) / CoFeB(0.6) / Pt(5.0) used for the carrier in the embodiment. Clearly, the magnetization of the thin film is out - of - plane, having a sharp coercive - force - induced magnetic switch to saturation. H C represents the coercive force field, i.e., the magnetic field required to magnetically switch the thin film to its saturated magnetic state.

[0251] FIG. 3(a) shows how the magnetic states of the barcode (or other 2 - D code) and carriers 30, 70 are linked to ensure that the code is always aligned with an external detector. First, as described above, before the carrier is released into the solution, H CA magnetic field exceeding this is used to set the magnetization of the carrier to the "up" state. In solution, if the carrier is not exposed to any strong magnetic field pulses, the carrier will retain this magnetization state. H C Any applied magnetic field less than this will simply cause the rotation of the carrier to align the magnetic moment M with the externally applied magnetic field. Typically, a field strength of 10 - 1000 Oe and a frequency of 0 - 50 Hz can be used to control the movement of the carrier. Since the barcode is fabricated on the top surface of each carrier, coated with 30 - 40 nm of Au and can be optically imaged through the Au, aligning the magnetic moment M with the external magnetic field corresponds to uniquely aligning the barcode face of the carrier in the direction of the applied magnetic field (H). Thus, as shown in Figure 3, the carrier may be oriented on a substrate, such as a planar substrate, by an applied magnetic field and may be read by any associated detector, such as a barcode reader and a fluorescence detector or camera.

[0252] Figure 3(b) shows an image of the carrier on a planar substrate and an image displaying a readable code such as a barcode.

[0253] Figure 4 shows the floating magnetic field strength as a function of the distance from the surface of carriers 30, 70 of the embodiment. Due to the high aspect ratio geometry of the carriers, it can be seen that the floating magnetic field is low. Advantageously, this reduces any tendency for the carriers to aggregate.

[0254] A carrier according to an embodiment of the present invention may be used to implement a multiplex assay as follows. The steps of the process are shown in FIGS. 5(a) and (b). For example, as described above, each carrier 100 is fabricated by lithography and patterned with a barcode 102 (preferably, a Quick Response (QR) code or a two-dimensional data matrix code). A predetermined barcode, or other readable code, is assigned to a desired assay sample, such as a specific protein to be identified in a multi-channel bioassay. Then, as shown in FIG. 5(a), each carrier carrying the code corresponding to a specific protein is functionalized with a capture antibody 104 specific to that respective protein. This can be carried out using conventional biochemical protocols. The gold surface of the carrier is suitable for this functionalization.

[0255] In this and other embodiments, if other functionalizations of the carrier are required, materials other than gold may be used on one or both of the top and bottom surfaces of the carrier. For example, SiO2 may be used.

[0256] Detection of the analyte is carried out with a conventional sandwich immunoassay. When the capture antibody captures the target protein 106, exposure of the magnetic carrier to a fluorescently labeled detection antibody 108 that is complementary to the capture antibody binds to and labels the protein. Then, as will be understood by those skilled in the art, the fluorescence of the fluorophore 110 in the detection antibody can be used to indicate that the protein has been captured and thus was present in the sample being tested in the assay.

[0257] Accordingly, a simple multiplex analyte capture platform may be provided for any desired application, including a plurality of sets (or groups) of magnetic carriers, each set of carriers carrying a unique code and being functionalized with a corresponding capture antibody. For a desired range of target proteins, a plurality of sets of carriers corresponding to those target proteins may be mixed together in an assay sample, such as a patient sample in which a diagnosis is to be performed using a multichannel assay.

[0258] In an assay according to a preferred embodiment, shown in FIG. 5(b), the analyte reagent is composed of a desired set 120 of functionalized magnetic carriers carried in a liquid medium 122. In a typical example, the analyte reagent may include a set or group of approximately 100 - 1000 coded magnetic carriers (MCs) functionalized with a capture antibody for each target protein 106. The analyte reagent is mixed with the sample to be analyzed and reacted with any target proteins present. The magnetic carriers are then removed from the sample and the liquid medium by magnetic separation 124. This includes using an external magnetic field 126 to attract the carriers together (e.g., as they gather at the bottom 128 of the container holding the sample), removing or decanting the sample. The carriers are then resuspended in a liquid medium 130 and exposed to a corresponding fluorescently labeled detection antibody 108. Then, via the application of an external magnetic field, the carriers are driven or induced to be placed on a reading surface 132. For example, the surface may be a slide glass. In particular, each carrier is magnetized out of the plane in a magnetization state in a unique direction towards or away from the upper surface of the carrier, so that the carriers can be induced such that all the carriers are in the same plane on the reading surface with respect to each other, and also such that all the carriers are similarly oriented, for example, with the upper surface of each carrier facing away from the reading surface.

[0259] In an alternative embodiment, the carriers may be magnetized in-plane in a magnetic field state parallel to the top or bottom surface of the carrier. The carriers can then be oriented by an external magnetic field on the reading surface, but it is not possible to align all the carriers with the top or bottom surface of each carrier facing away from the surface. This obvious problem can be solved in one of two ways. Since the carriers can be made with readable information on both the top and bottom surfaces of the carriers, the information can be read from either surface. Alternatively, the carriers may be made such that, for example, the layer of carriers is made sufficiently transparent so that information can be read from both the top and bottom surfaces.

[0260] When the carriers are placed on the reading surface 132, two images of the carriers can be taken using a suitable camera and control software. The first image 134 is a bright-field image showing the code or information on each carrier. This unambiguously identifies which carriers in the image carry the capture antibody for each target protein, or in other words, which channel of the multi-channel assay each carrier belongs to. The second image 136 is a fluorescence image of the carriers. If the carrier emits fluorescence, the detection antibody on that carrier has captured the corresponding protein, and the fluorescence intensity can indicate the concentration of the protein in the sample. If the carrier does not emit fluorescence, then that carrier has not captured its corresponding protein and is not present in the sample. Thus, overlaying the two images can identify which proteins were present in the sample by assigning a fluorescence intensity value to each carrier. The corresponding analysis software then indicates which proteins are present in the sample and their concentrations.

[0261] An important feature of multi-channel analysis enabled by carrier barcoding is that the potential number of plex channels is very large, up to the maximum number of channels that can be encoded by the barcode, which may even be 1000 channels or more. At the same time, the carriers within an individual channel can be uniquely identified, achieving little or no crosstalk between channels. In comparison, conventional bead-based bioassays use a fluorescence-based channel identification system that is much less resistant to crosstalk. For example, one prior art system uses the ratio of fluorophores on barcoding beads and fluorophore-labeled antibodies as positive signaling for analyte detection. This poses challenges to the reliability of channel identification and significantly limits the plex number.

[0262] (PCT / GB2019 / 053188: Clause describing priority features) 1. A magnetic carrier comprising a layer structure between the upper surface of the carrier and the opposing bottom surface of the carrier, the layer comprising one or more magnetization layers, wherein the ratio of the lateral dimension of the one or more magnetization layers to the thickness or collective thickness of the one or more magnetization layers is greater than 500.

[0263] 2. The magnetic carrier according to clause 1, wherein the layer comprises a non-magnetic layer.

[0264] 3. The magnetic carrier according to clause 1 or 2, wherein the ratio of the lateral dimension of the one or more magnetization layers to the thickness or collective thickness of the one or more magnetization layers is greater than 1000, preferably greater than 2000.

[0265] 4. The one or more magnetization layers comprise a volume V of a magnetic material having a magnetization or average magnetization Ms, and the cross-section of the one or more layers has an aspect ratio AR, and AR / Ms 2 (Ms is measured in A / m) is 8 * 10 -10 (A / m) -2A magnetic carrier according to any one of clauses 1 to 3, which is larger than...

[0266] 5. The one or more magnetization layers include a volume V of a magnetic material having a magnetization or an average magnetization Ms, and a cross-section of the one or more layers has an aspect ratio AR, and AR / Ms (where Ms is measured in A / m) is larger than 0.001 (A / m). The magnetic carrier according to any one of clauses 1 to 4.

[0267] 6. The upper and bottom surfaces of the carrier are separated by the thickness of the carrier between 5 nm and 200 μm, and / or the minimum lateral dimension of the carrier is larger than 1 μm, preferably larger than 5 μm or 10 μm. The magnetic carrier according to any one of clauses 1 to 5.

[0268] 7. The ratio of the minimum lateral dimension of the carrier to the thickness of the carrier is larger than 10. The magnetic carrier according to any one of clauses 1 to 6.

[0269] 8. The maximum lateral dimension of the carrier is less than 1000 μm, preferably less than 500 μm or 200 μm. The magnetic carrier according to any one of clauses 1 to 7.

[0270] 9. The minimum lateral dimension of the carrier is at least 10% of the maximum lateral dimension of the carrier, preferably at least 30% or 50% or 70% of the maximum lateral dimension. The magnetic carrier according to clause 8.

[0271] 10. The lateral peripheral portion of the carrier has a shape including convex or straight side surfaces, preferably a shape having no convex side surfaces and / or recessed corners. The magnetic carrier according to any one of clauses 1 to 9.

[0272] 11. The lateral dimension of the magnetic layer or at least one of the magnetic layers is the same as the lateral dimension of the carrier. The magnetic carrier according to any one of clauses 1 to 10.

[0273] 12. The non-magnetic layer of the magnetic carrier according to any one of clauses 1 to 11 contains a material selected from non-magnetic metals, non-metals, semi-metals and compounds, AI, Ta, Pt, Pd, Ru, Au, Cu, W, MgO, Cr, Ti, Si, Ir, SiO2, SiO, Sn, Ag, SiN, Ge, polymers, plastics, alloys of these materials, and composites or mixtures of these materials.

[0274] 13. Each of the magnetization layers or each of the magnetization layers of the magnetic carrier according to any one of clauses 1 to 12 contains a material selected from magnetic metals, magnetic alloys, magnetic compounds and superparamagnetic nanocarrier composites, such as Fe, Co, Ni, CoFe, CoFeB, FePt, CoNi, NiFe and Fe2O3.

[0275] 14. Each of the magnetization layers or each of the magnetization layers of the magnetic carrier according to any one of clauses 1 to 13 is an out-of-plane magnetization layer.

[0276] 15. Each of the magnetization layers or each of the magnetization layers of the magnetic carrier according to any one of clauses 1 to 13 is an in-plane magnetization layer.

[0277] 16. Each of the magnetization layers or each of the magnetization layers of the magnetic carrier according to any one of clauses 1 to 15 is disposed between the layer of non-magnetic material and the second layer of non-magnetic material.

[0278] 17. Each of the magnetization layers or each of the magnetization layers of the magnetic carrier according to clause 16 is separated from the upper surface of the carrier by more than 25% of the thickness of the carrier and separated from the bottom surface of the carrier by more than 25% of the thickness of the carrier.

[0279] 18. Each of the magnetization layers or each of the magnetization layers of the magnetic carrier according to any one of clauses 1 to 17 has a thickness greater than 0.1 nm, preferably greater than 0.5 nm.

[0280] 19. The collective thickness of the one or more magnetization layers is less than 25% of the thickness of the carrier, preferably less than 15% or 10%, of the magnetic carrier according to any one of clauses 1 to 18.

[0281] 20. Each of the magnetization layers or the magnetization layer is a thin film multilayer, of the magnetic carrier according to any one of clauses 1 to 19.

[0282] 21. The net magnetic field (floating magnetic field) averaged across the upper or lower surface of the carrier is less than 2500 A / m, preferably less than 800 A / m or 400 A / m, of the magnetic carrier according to any one of clauses 1 to 20.

[0283] 22. The magnetic carrier according to any one of clauses 1 to 21, produced by lithography.

[0284] 23. The carrier carries readable information such as a readable code selected from a barcode or a two-dimensional code, and is readable on one or both of the upper or lower surfaces of the carrier, or from one or both of them, of the magnetic carrier according to any one of clauses 1 to 22.

[0285] 24. The surface of the carrier is functionalized, and the readable information corresponds to the functionality of the carrier, of the magnetic carrier according to clause 23.

[0286] 25. The upper or lower surface of the carrier is functionalized, the information is readable on or from the same surface that is functionalized, and / or the surface of each carrier is functionalized and each carrier holds the readable information corresponding to the functionalization of the carrier, of the magnetic carrier according to clause 24.

[0287] 26. A method for manufacturing the magnetic carrier according to any one of clauses 1 to 25 by a lithography process.

[0288] 27. A method for performing an assay, comprising providing a carrier according to clause 24 or 25 to a liquid assay sample, interacting the functionalization of the carrier with the assay sample, applying a magnetic field to induce the carrier to a reading position, and obtaining an assay result by reading the readable information and the interaction between the functionalization of the carrier and the assay sample.

[0289] 28. A method for performing a multi-channel assay, comprising providing a plurality of carriers according to clause 24 or 25 to an assay sample, interacting the functionalization of the carriers with the assay sample, applying a magnetic field to induce the carriers to a reading position, and obtaining an assay result by reading the readable information of two or more carriers and the interaction between the corresponding functionalization of the carriers and the assay sample.

Claims

1. A carrier system for an assay comprising a carrier fixed to a substrate by a release layer, wherein the carrier is suitable for receiving an assay sample, and the release layer is configured to release the carrier from the substrate in the presence of a biocompatible aqueous solution during use, while the carrier is in contact with the substrate, is adapted to receive the assay sample on the carrier, and / or, the assay sample is suspended in the biocompatible aqueous solution, and when the carrier system is in contact with the biocompatible aqueous solution, the assay sample is received on the carrier, and the carrier system is configured such that the carrier is released from the substrate. Carrier system.

2. The carrier system according to claim 1, wherein the release layer is configured such that the biocompatible aqueous solution maintains biocompatibility after activation of the release layer, and / or, the release layer comprises a material that is water-activatable, and / or, the release layer is not activatable in a non-aqueous solvent, and / or, the release layer comprises at least one of sugar, or polyvinyl alcohol, or poly(acrylic acid), or poly(lactic-co-glycolic acid). Carrier system.

3. The carrier system according to claim 1 or 2, wherein the carrier comprises a magnetic material. Carrier system.

4. The carrier system according to claim 3, wherein the carrier comprises a layer structure between the top surface of the carrier and the opposing bottom surface of the carrier, and the layer comprises one or more magnetization layers. Carrier system.

5. The carrier system according to claim 4, wherein the ratio of the lateral dimension of the one or more magnetization layers to the thickness or collective thickness of the one or more magnetization layers is greater than 500, and / or, the minimum lateral dimension of the carrier is between 5 micrometers and 200 micrometers. Carrier system.

6. The carrier system according to claim 1 or 2, wherein the carrier is defined by lithography, and / or, the carrier comprises a photoresist layer, and / or, the surface of the carrier is adapted to receive the assay sample, and the surface comprises a gold cap layer to which a polymer is covalently bonded by a thiol group. Carrier system.

7. A carrier system according to claim 1 or 2, wherein the carrier includes a readable code selected from a barcode or a two-dimensional code, and / or, includes a plurality of carriers, each of the carriers being fixed to the substrate by the release layer, and / or, a sterile package in which the carrier fixed to the substrate is removable for use, the carrier system.

8. A method of manufacturing a carrier system for an assay, comprising the step of providing a substrate, the step of forming a release layer on the substrate, the step of depositing a carrier for receiving an assay sample on the release layer such that the carrier is fixed to the substrate, comprising, the release layer being configured to release the carrier from the substrate in the presence of a biocompatible aqueous solution during use, the carrier being adapted to receive the assay sample on the carrier while the carrier is in contact with the substrate, and / or, the assay sample being suspended in the biocompatible aqueous solution, and when the carrier system is in contact with the biocompatible aqueous solution, the assay sample is received on the carrier and the carrier system is configured such that the carrier is released from the substrate, the method.

9. A method according to claim 8, wherein the step of forming the release layer on the substrate includes spin-coating the release layer, and / or, the step of depositing the carrier on the release layer includes creating the carrier on the release layer, and / or, the release layer is adapted to release the carrier from the substrate in the presence of the biocompatible aqueous solution within a time period between 1 hour and 72 hours during use, the method.

10. A method according to claim 8 or 9, further comprising the step of adapting the surface of the carrier such that the surface is suitable for receiving the assay sample.

11. A method according to claim 8 or 9, further comprising the step of sterilizing the carrier system by immersing the carrier system in ethanol after the step of depositing the carrier on the release layer, and / or, Further comprising the step of packaging the carrier system in a sterile package with the carrier fixed to the substrate, and / or, The method of depositing the carrier includes forming a magnetic structure.

12. A method of performing an assay using the carrier system according to claim 1 or 2, comprising: Introducing a biocompatible aqueous solution into the carrier system to detach the carrier; and Introducing the sample for the assay into the carrier such that the sample is received by the carrier. The method wherein the sample is received by the carrier while the carrier is in contact with the substrate.

13. The method according to claim 12, wherein the assay sample is suspended in the biocompatible aqueous solution, and / or the carrier contains a magnetic material, and the method further includes applying a magnetic field to the carrier, the magnetic field acting to hold the carrier in contact with the substrate even after the release layer has detached the carrier from the substrate.

14. The method according to claim 13, wherein the magnetic field is applied to hold the carrier in contact with the substrate for at least 5 seconds, or at least 1 minute, or at least 5 minutes, or at least 30 minutes.

15. A method of using the carrier system according to claim 1 or 2, comprising: introducing the sample for the assay into the carrier such that the sample is received by the carrier while the carrier is in contact with the substrate; and storing the sample received on the carrier.

16. The method according to claim 15, wherein the method further includes freezing the sample received on the carrier for storage.

17. The method according to claim 15, further comprising detaching the carrier from the substrate before storing the sample received on the carrier.

18. A method for performing an assay, comprising: providing the carrier fixed to the substrate by a release layer configured to detach the carrier from the substrate in the presence of a biocompatible aqueous solution; and contacting the carrier fixed to the substrate with a biocompatible aqueous solution in which the assay sample is suspended. Receiving the assay sample on the carrier while the carrier is in contact with the substrate; Detaching the carrier from the substrate by activation of the release layer with the biocompatible aqueous solution; A method comprising.

19. The carrier system according to claim 1 or 2, wherein the assay sample comprises adherent cells.

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