Carrier, carrier system and method

Abstract

A carrier (100) for an adhesion assay sample is provided, the carrier comprising a sample face (25) for receiving an adhesion assay sample, and a peripheral region (28) between the sample face and an edge of the carrier, the peripheral region including a sample adhesion reducing coating such that, in use, the adhesion assay sample adheres less strongly to the peripheral region than to the sample face.

Description

[Technical Field]

[0001] The present invention relates to carriers, carrier systems and methods, and in particular to carrier systems for assays and methods for producing carrier systems. [Background technology]

[0002] An assay is a research technique for qualitatively or quantitatively determining 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 the cell or cell culture, although assays may more generally involve metabolites, peptides, proteins, nucleic acids, extracellular vesicles, organelles, cells, or tissues.

[0003] Some biological assay samples, such as certain types of cells, can be suspended in aqueous solutions. 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. Assays for such samples, including measurement steps, must typically be performed while the assay sample is attached to the bottom of a well plate. Unfortunately, in such assays, the assay process can be 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 from the well plate. This can result in sample damage.

[0004] An improved approach for performing assays using adherent assay samples, such as adherent cells, is to use carriers or particles to carry the assay sample. Examples of such carrier systems are described in U.S. Patent Nos. 5,629,999 and 5,729,999.

[0005] In such systems, a plurality of carriers can be provided immobilized on a substrate, and adherent cells can be received on the exposed surface of the carrier, e.g., from a biocompatible solution. The carriers can be magnetic. In this case, the carriers can be held on the substrate (by an applied magnetic field) to allow time for the sample to adhere to the carrier. The magnetic carriers, along with the adherent sample, can then be detached from the substrate and directed or manipulated by the applied magnetic field throughout the assay.

[0006] However, when an adhesive sample is received on a carrier, problems arise, particularly when multiple carriers are placed in close proximity to one another on a substrate, in that the sample may adhere or migrate to the substrate, or the sample may adhere to both the substrate and the carrier.

[0007] Approaches for spatially restricting cell growth are known for specific applications.

[0008] Conventionally, cells can be grown or cultured on poly(dimethylsiloxane) (PDMS) substrates, as in Non-Patent Document 1. This method does not require the use of a carrier, but describes a method for spatial control and patterning of cell growth on PDMS substrates. In this method, patterned barriers to cell adhesion are formed on the substrate using light-activated graft polymerization of polyethylene glycol, diacrylic acid (PEG-DA). This promotes cell growth within the microchannels defined between the barriers.

[0009] Another approach is described in Non-Patent Document 2. Wang et al. fabricated an array of microwells on a glass substrate. Each microwell contained a planar SU-8 micropallet at its base. Adherent cells could be accommodated within the microwells and on the micropallets, and then individual micropallets could be expelled from those microwells by a focused laser pulse directed through the glass substrate below any desired micropallet. The cells carried by the detached micropallets could then be recovered, cultured, and clonally propagated. Wang et al. described two approaches to forming microwells. One was to form virtual air walls between the micropallets by applying a hydrophobic coating to the glass substrate and forming air bubbles that adhered to the substrate by Cassie-Baxter wetting. The other, similar to Moustafa et al., formed photopolymerized PEG hydrogel walls on a glass substrate, but defined PEG walls between the micropallets by exploiting the difference in UV transmittance between the glass substrate and the SU-8 micropallets to localize photopolymerization. By using focused laser pulses, individual micropallets could be pulled away from between the walls on the substrate. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2020 / 099846 [Patent Document 2] International Publication No. 2021 / 224631 [Non-patent literature]

[0011] [Non-Patent Document 1] Large area micropatterning of cells on polydimethylsiloxane surfaces(Moustafa et al, Journal of Biological Engineering 2014, 8:24) [Non-patent document 2] Micropallet arrays with poly(ethylene glycol) walls(Wang et al, in The Royal Society of Chemistry's Lab Chip, 2008, 8, 734-740) Summary of the Invention [Problem to be solved by the invention]

[0012] However, when magnetically manipulated carriers are used, as in Patent Documents 1 and 2, several problems arise when these approaches are used to prevent cell attachment to substrates. One is that the presence of walls between the carriers on the substrate can prevent the carriers from being detached from the substrate. Wang et al. used laser pulses to actively drive their micropallets from the substrate. However, the carriers, i.e., the micropallets, may be held in place by the walls if they are simply detached from the substrate by removing the applied magnetic field. Another problem is that Wang et al.'s approach requires the use of a transparent glass substrate both to provide a photomask to enable the formation of the walls and to enable the detachment of the micropallets using laser pulses.

[0013] A further problem with the conventional use of carriers for adhesion assay samples arises when magnetic carriers are used and the carriers are directed or manipulated through the assay by an applied magnetic field. Under these circumstances, adhesion assay samples can be subjected to significant shear forces as the sample is driven through the assay medium (typically a biocompatible solution). While this is not a problem for many assay samples, for lower levels of adhesion and larger assay samples, shear forces can be detrimental to the results of the assay.

[0014] The object of the present invention is to solve these problems. [Means for solving the problem]

[0015] Advantageously, the present invention provides a carrier, a carrier system and a method for adhesion assay samples as defined in the accompanying independent claims, to which reference is now made. Preferred or advantageous features of the invention are set out in the dependent subclaims.

[0016] Thus, in a first aspect, the present invention can provide a carrier for an adhesion assay sample. The carrier may include a sample surface for receiving an adhesion sample and a peripheral region surrounding the sample surface and disposed between the sample surface and the edge of the carrier. At least a portion of the carrier includes one or more coatings such that the adhesion sample adheres less strongly to the peripheral region than to the sample surface. Suitable coatings for achieving this may be applied to the sample surface or the peripheral region, or to both the sample surface and the peripheral region. Preferably, the peripheral region includes a sample adhesion-reducing coating. Advantageously, the presence of the coating(s) on the carrier may promote adhesion of the adhesion sample to the sample surface. The sample surface forms a defined region that is spaced apart or separated from the edge of the carrier.

[0017] Advantageously, the coating(s) may ensure that, in use, the adhered sample adheres more strongly to the sample surface than to the surrounding areas. Advantageously, this may mean that the sample is only adhered to a defined area of ​​the carrier surface, making it easier to analyze the carrier to which the sample is adhered. For example, it may be easier to analyze a sample adhered to the central sample surface of the carrier, which is easily visible from above, rather than a sample adhered to the periphery or edge of the carrier.

[0018] The surface of the carrier, including the sample surface and the surrounding area, is preferably flat or planar or nearly flat or planar. For example, the body or base layer of the carrier may include a flat or planar surface for carrying the sample, and may then be subjected to a surface treatment or coating to increase or promote sample adhesion to the sample surface and / or to reduce or prevent sample adhesion to the surrounding area.

[0019] Surface treatments such as coatings on the sample surface and / or the surrounding area may have different thicknesses, but within the scope of embodiments of the present invention the resulting surface may be considered to be a substantially flat or planar surface. In other words, any height difference between the sample surface and the surrounding area is preferably less than 50% of the maximum thickness of the coated carrier, particularly preferably less than 20% or 10% of the maximum thickness of the coated carrier.

[0020] The peripheral region may be raised relative to the sample surface. When the peripheral region is raised relative to the sample surface, the raised peripheral region can further provide a physical obstacle to cells if they tend to detach from the sample surface. This physical obstacle can enhance the effectiveness of the sample adhesion-reducing coating in retaining cells on the sample surface. For example, the peripheral region may then be referred to as or include a wall for confining or surrounding cells adhering to the sample surface. The carrier may have a wall between the sample surface and the edge of the carrier to promote adhesion of the adhered sample to the sample surface and to prevent the adhered sample from detaching from the sample surface. To achieve this, the geometry of the wall may be sufficient to prevent sample movement or adhesion on surfaces other than the sample surface, which may also be advantageous if, in use, the adhered sample adheres more weakly to the wall than to the sample surface.

[0021] Advantageously, the carrier may be placed or positionable on a substrate to receive an adhesion assay sample, in which case a peripheral region, e.g. a wall, is preferably between the sample surface and the substrate when the carrier is placed on the substrate.

[0022] The peripheral region preferably completely surrounds the sample surface in a continuous loop or ring around the periphery of the carrier, preferably extending from the carrier edge to the periphery of the sample surface. Particularly preferably, in embodiments comprising a wall, the wall surrounds or completely encircles the sample surface.

[0023] Advantageously, the coating is applied to the peripheral region of the carrier, and the coating is configured to reduce or prevent sample adhesion to the peripheral region, such that the adhered sample adheres less strongly to the peripheral region than to the sample surface. Thus, the coating for the peripheral region may be referred to as a sample repellent coating, a non-adhesion coating, or an adhesion-reducing coating. Since the sample is typically some type of biological cell, the coating is preferably a coating that reduces cell adhesion to the peripheral region of the carrier.

[0024] The coating applied to the peripheral region may be a non-ionic surfactant. In a preferred embodiment, the peripheral region may be coated with one or more of Pluronic® F-127, or Poly-HEMA, or other poloxamers.

[0025] The base or body of the carrier to which the coating is applied may be fabricated by any convenient method. In a preferred embodiment, the base is fabricated lithographically. In such a case, the base, including its peripheral boundary below the peripheral region, may comprise a photoresist such as SU-8. A central portion of the carrier's base may then be selectively treated or coated with, for example, a metal layer (e.g., a magnetic layer or a gold layer) using a second lithography step.

[0026] Alternatively, the base or body of the carrier may be formed using different materials in its central portion below the sample surface and its peripheral portion below the peripheral region.

[0027] If the peripheral area of ​​the carrier contains a photoresist material such as SU-8, the peripheral area is oxygen plasma treated and then coated with silane-terminated PEG molecules, so that the silane covalently bonds to the O2 plasma-treated surface and the PEG molecules form an anti-adhesion coating.

[0028] Advantageously, the provision of a non-sample-adherent peripheral region can reduce the likelihood of sample cell transfer between nearby carriers during use, because the peripheral region can create spatial separation between the sample surfaces of adjacent carriers when the carriers are placed on a substrate. When multiple carriers are placed on top of each other on a substrate, such that the edge of one carrier overlaps the edge of another carrier (which can occur, for example, when multiple carriers are deposited or magnetically manipulated on a substrate during an assay), the peripheral region also improves the likelihood that the sample surface of each carrier will remain visible from above during analysis.

[0029] In embodiments of the present invention that include walls, the geometry of the walls may help to restrain the adhered sample to the sample surface, but the geometrical effect of the walls themselves is unlikely to be significant. Therefore, advantageously, at least a portion of the surface of the wall may include a surface treatment, such as a coating, e.g., a sample adhesion-reducing coating, that causes the adhered sample to adhere less strongly to the wall than to the sample surface. For example, the wall or a portion of the wall may be coated with a nonionic surfactant. The wall or a portion of the wall may be coated with Pluronic F-127, Poly-HEMA, or other poloxamers. If the wall comprises a photoresist material such as SU-8, the wall may be oxygen plasma treated and then coated with silane-terminated PEG molecules, so that the silane covalently bonds to the O2 plasma-treated surface and the PEG molecules form an anti-adhesion coating.

[0030] Instead of or in addition to providing a sample adhesion-reducing coating on the peripheral region, the sample surface may include a coating configured to cause the adhered sample to adhere more strongly to the sample surface than to the peripheral region. This may help to restrain the adhered sample to the sample surface. Such a sample surface coating may be referred to as an adhesion-promoting coating. For example, the sample surface may include a biofunctionalized coating configured to attach or adhere to the assay sample. The sample surface may also include a charged coating. This is particularly preferred when the assay sample is naturally charged. For example, cells typically have a membrane potential of minus 40 to minus 80 millivolts. The provision of a coating defining a positively charged surface can promote the attachment of the assay sample to the charged surface of the carrier. It has been found that providing a charged surface, preferably a positively charged surface, can advantageously result in more effective cell adhesion. The coating may include a charged polymer.

[0031] In some embodiments, the sample surface may include a gold layer in the form of a gold cap, and the adhesion-promoting coating may be applied over the gold layer.

[0032] The gold cap may be formed only on the area of ​​the carrier that will form the sample surface. If an adhesion-promoting coating is then applied over the gold layer, only the sample surface of the carrier will be biofunctionalized, and therefore only the sample surface of the carrier will tend to accept the assay sample, while the surrounding area (not including the functionalized gold cap) will not accept the assay sample.

[0033] The polymer (charged polymer) may be covalently bonded to a gold cap forming the sample surface. The sample surface adapted to receive the assay sample may include a gold cap layer to which the polymer is covalently bonded via thiol groups. This advantageously ensures that each polymer adsorbed on the surface has the same orientation. Alternatively, the polymer may be adsorbed to the gold cap of the carrier by van der Waals forces.

[0034] 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.

[0035] Alternatively or additionally, the coating on the sample surface may include multiple ligands. The multiple ligands may include antibodies. If the assay sample of the assay is a cell, the multiple ligands may include antibodies that specifically bind to a cellular receptor, such as an integrin.

[0036] If the assay sample is a cell, the sample surface coating may alternatively or additionally comprise 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.

[0037] In a preferred embodiment, the sample surface may be substantially flat and preferably parallel to the substrate when the carrier is placed on it. Advantageously, the or each carrier may comprise a body or base, which is preferably laminar and may be of a high-aspect-ratio shape, so that its lateral dimensions, i.e., its length and width, may be many times greater than its thickness, for example, 500 or 1000 times greater than its thickness. The upper surface of the base (e.g., one of the larger surfaces in a high-aspect-ratio shape) may then form or carry the sample surface. A peripheral region may also preferably be provided on the upper surface of the base, and is provided in approximately the same plane (or coplanar) as the sample surface. Alternatively, a wall may extend upward from the upper surface of the base, adjacent to or spaced from the sample surface. As mentioned above, the sample surface and the peripheral region may carry surface treatments, such as coatings of different thicknesses, so that the sample surface and the peripheral region do not have to be exactly coplanar.

[0038] The carrier is preferably a planar particle. The carrier particle preferably includes a flat first surface and a flat second surface arranged parallel to the first surface on the opposite side of the carrier particle. The sample surface and the peripheral area are preferably provided on the same surface of the carrier, for example, both on the first surface, while the other surface may be attached, for example, to a release layer. Due to the presence of the peripheral area, the sample surface may not occupy the entire surface of the carrier, and the sample surface may not extend to the edge of the carrier.

[0039] To achieve the desired effect of confining the sample away from the carrier edge and preventing sample migration to other carriers, at least a portion of the peripheral region preferably has a width greater than 1 micrometer and less than 50 micrometers, or greater than 5 micrometers and less than 30 micrometers, preferably greater than 8 micrometers and less than 25 micrometers. For example, the peripheral region may have a width of 10 micrometers. Preferably, the peripheral region has a uniform width around the sample surface.

[0040] To achieve the desired geometric effect of the walls, at least a portion of the walls may have a height relative to the sample plane that is greater than 1 micrometer, greater than 2 micrometers, or greater than 3 micrometers, and less than 50 micrometers, less than 20 micrometers, or less than 15 micrometers, and at least a portion of the walls may have a lateral thickness or maximum lateral thickness that is greater than 1 micrometer and less than 50 micrometers, preferably greater than 5 micrometers and less than 40 micrometers.

[0041] To modify the effectiveness of the wall to restrain or contain the adherent sample, at least a portion of the wall may have a rectangular cross-section, or a flared or rounded cross-section. For example, the cross-section of at least a portion of the wall may be stepped, or its thickness may vary non-monotonically. In particular, the cross-section of the wall may be flared so that the cross-section of the wall away from the sample surface is larger than the cross-section of the wall closest to the sample surface. For example, a portion of the wall may overhang the sample surface. This can improve containment of the adherent sample. Alternatively, the cross-section of the wall may be flared so that the cross-section of the wall away from the sample surface is smaller than the cross-section of the wall closest to the sample surface. This allows adherent cells to more easily reach the sample surface of the base. The wall preferably has the same cross-section throughout its length.

[0042] Flared walls, where the top is thicker than the base of the wall, or walls that overhang the sample surface, can advantageously provide a mechanical barrier to cell migration away from the sample surface. Alternatively, flared walls, where the top is less thick than the base of the wall, can advantageously allow any cells that may adhere to the wall to migrate to the sample surface. Thus, wall shapes may be optimized for different cell or sample types, as well as different wall and sample surface coatings.

[0043] Advantageously, the carrier may have lateral dimensions, preferably a minimum lateral dimension, greater than 5 micrometers and less than 300 micrometers, or preferably greater than 10 micrometers and less than 200 micrometers.

[0044] The portion of the carrier below the peripheral region may be manufactured by any suitable method, but may advantageously, for convenience and efficiency, comprise one or more materials that are also present in the portion of the carrier below the sample surface. The portion of the carrier below the peripheral region is preferably manufactured at the same time as, or in the same process as, the portion of the base below the sample surface. These portions of the carrier base may be of the same material(s) that extend continuously below both the peripheral region and the sample surface.

[0045] For example, the base portion may conveniently be manufactured using a lithographic process, in which case the magnetic material may also be incorporated into the carrier.

[0046] In a preferred embodiment, the peripheral region is simply a coated region on the base portion of the carrier.

[0047] The peripheral region of the carrier may be fabricated using nanoimprint lithography or microimprint lithography.

[0048] In a preferred embodiment, the sample surface is simply a coated area on the base of the carrier.

[0049] In one embodiment, particularly if the carrier is fabricated using photolithography, the carrier base at least below the peripheral region may comprise a structural photopolymer, preferably photoresist, SU-8 or AZ-10nXT. Alternatively or additionally, the base at least below the peripheral region may comprise a polymer such as a thermoplastic polymer, preferably PDMS.

[0050] The walls of the carrier may be manufactured by any suitable method, but advantageously, for convenience and efficiency, the walls may comprise material that is also present in the base portion, and the walls are preferably manufactured using a process that is also used to manufacture the base portion. For example, the walls and base portion may conveniently be manufactured using a lithographic process, in which case magnetic material may also be incorporated into the carrier.

[0051] The walls may be fabricated using nanoimprint lithography or microimprint lithography.

[0052] In one embodiment, particularly if the carrier is fabricated using photolithography, the walls may comprise a structural photopolymer, preferably photoresist, SU-8 or AZ-10nXT. Alternatively or additionally, the walls may comprise a polymer such as a thermoplastic polymer, preferably PDMS.

[0053] In a further aspect, the present invention can advantageously provide a carrier system for assays, comprising a plurality of carriers. In the carrier system, the carriers are preferably first provided fixed to a substrate, for example, by a release layer. The carriers on the substrate may then be exposed to a biocompatible solution in which an adhesion assay sample (e.g., adherent cells) is suspended, thereby allowing the adhesion sample to adhere to the sample surface of the carrier. The release layer may be activated by or dissolve in the biocompatible solution, resulting in the carrier carrying the adhesion sample being released from the substrate into the solution. Advantageously, during the assay, the adhesion sample may be supported on the sample surface of each carrier and may be prevented from bridging or crossing from the sample surface to the substrate by a peripheral region, e.g., a wall, separating the sample surface from the edge of the carrier.

[0054] In a further preferred embodiment, the substrate between the carriers may additionally be subjected to a surface treatment or coating, which reduces adhesion to the adhesive sample and thus further reduces any risk that the sample may bridge or cross over from the carrier to the substrate.

[0055] For example, during manufacture of the carrier system, a release layer is preferably applied to the entire substrate, including the area between the (subsequently formed) carriers. In one embodiment, the release layer between the carriers may be removed after at least the carrier base portion is formed, and the underlying substrate is treated or coated to reduce cell adhesion.

[0056] It may not be appropriate to apply such surface treatments or coatings to the release layer itself, as the release layer typically dissolves in the biocompatible solution as the adhesive sample is loaded onto the sample side of the carrier.

[0057] The carrier may advantageously be magnetic or may include a magnetic material, in which case it will be held on the substrate by an applied magnetic field even after the release layer has dissolved, thereby allowing the adhered sample to adhere to the sample surface for a longer period of time.

[0058] In yet another aspect, the present invention advantageously provides a method for performing an assay using the above-described carrier or carrier system. The method may advantageously include the steps of providing a plurality of carriers arranged on a substrate, introducing a biocompatible aqueous solution carrying an adherent sample to the carrier and the substrate so that the adherent sample adheres to the sample surface of the carrier but not to the peripheral region of the carrier, and peeling the carrier from the substrate. The carrier may be held in place on the substrate by a release layer between the carrier and the substrate, which release layer is activated by the biocompatible aqueous solution to peel the carrier. Additionally or alternatively, the carrier may be magnetic or include a magnetic material and may then be held in place on the substrate by an applied magnetic field and peeled from the substrate by changing or removing the applied magnetic field.

[0059] In a further aspect of the present invention, it should be noted that the carriers of the present invention are preferably magnetically operable, for example, during the assay stage. Thus, after detachment from the substrate, the solution during the assay can contain multiple substrates and carry adhesive samples. While in the solution, the carriers may move closer to each other, and it is important to avoid the adhesive sample from transferring from one carrier to another. The sample-repellent effect of the peripheral area surrounding the adhesive sample on each carrier can advantageously help keep the sample surfaces of different carriers spaced apart from each other when the carriers are in the solution, thus reducing the risk of the adhesive sample transferring from one carrier to another.

[0060] In some cases, individual carriers may include a readable code or may otherwise be marked to allow individual carriers or groups of carriers to be identified in the assay. When adherent samples are placed on individual carriers at the start of the assay, it is essential that the samples do not transfer from one carrier to another. Because carriers in solution may be close to or pass through other carriers, a coated peripheral area surrounding the sample on the carrier in solution can advantageously prevent sample transfer between carriers.

[0061] A readable code, such as a barcode or QR code, may be applied to the surface of the carrier, for example, or may comprise one or more holes defined through or in the base of the carrier. Preferably, the code may be located in the peripheral region. Advantageously, this prevents the code from reducing the area of ​​the sample surface available for cell attachment. Preferably, the wall comprises the readable code. Advantageously, a readable code included in the wall prevents the code from reducing the area of ​​the sample surface available for cell attachment. The code may partially penetrate the wall. The code may penetrate through the wall to the carrier base. The readable code may comprise a plurality of dots, dashes, or dots and dashes. The code may comprise several lines. The code may be formed by lithography.

[0062] The readable code can take various forms as a marking in the carrier that encodes information that identifies the carrier on which it is applied. When imaged, the pattern in a given carrier can be analyzed for its coded information and therefore used to identify that particular carrier. The code is preferably readable by image analysis of a microscopic image.

[0063] Also in the assay, the magnetically manipulable carrier may generally be manipulated onto a substrate, e.g., for imaging or analysis. When the carrier is positioned on the substrate in this manner, the coated peripheral region of the carrier can advantageously prevent the transfer of adherent sample from the carrier to the substrate or from one carrier to another.

[0064] As noted above, the sample surface and peripheral region of a carrier embodying the invention may be coplanar, or optionally may not be coplanar. In one option, the peripheral region is raised relative to the sample surface. This aspect of the invention may advantageously relate to further aspects of the invention discussed below.

[0065] Thus, in this further aspect, the present invention may provide a carrier for an adhesion assay sample. The carrier may comprise a sample surface for receiving the adhesion sample, and a wall between the sample surface and an edge of the carrier for promoting adhesion of sample particles to the sample surface and preventing sample particles from moving away from the sample surface. To achieve this, the geometry of the wall may be sufficient to prevent sample migration or adhesion on surfaces other than the sample surface, but may also be advantageous if, in use, the adhesion sample adheres more strongly to the sample surface than to the wall.

[0066] The carrier may advantageously be positionable on a substrate for receiving an adhesion assay sample, in which case the wall is preferably between the sample surface and the substrate when the carrier is positioned on the substrate.

[0067] Particularly preferably, the wall surrounds or completely surrounds the sample surface.

[0068] In a preferred embodiment, the carrier may form part of a carrier system in which multiple carriers are provided on a substrate and preferably fixed to the substrate, for example, by a release layer. The carriers on the substrate may then be exposed to a biocompatible solution in which an adhesion assay sample (e.g., adherent cells) is suspended, thereby allowing the adhesion sample to adhere to the sample surface of the carrier. The release layer may be activated by the biocompatible solution or may dissolve in the biocompatible solution, resulting in the carrier carrying the adhesion sample being released from the substrate into the solution. Advantageously, the adhesion sample may be supported on the sample surface of each carrier and may be prevented from bridging or crossing from the sample surface to the substrate by a wall.

[0069] Advantageously, the carrier may be magnetic or may comprise a magnetic material, in which case the carrier is held on the substrate by an applied magnetic field even after the release layer has dissolved, thereby allowing the adhered sample to adhere to the sample surface for a longer period of time.

[0070] In preferred embodiments, the sample surface is substantially flat, preferably parallel to the substrate when the carrier is placed on it. Advantageously, the or each carrier may comprise a base portion, which is preferably laminar and may be a high aspect ratio shape so that its lateral dimensions (length and width) may be many times greater than its thickness, for example 500 or 1000 times greater than its thickness. An upper surface of the base portion (e.g. one of the larger surfaces of the high aspect ratio shape) may then form or carry the sample surface, and a wall may extend upwardly from the upper surface of the base portion so as to be adjacent to or spaced from the sample surface.

[0071] While the geometric shape of the walls may be sufficient to constrain the adhered sample to the sample surface, for some types of adhered samples, the geometric effect may not be sufficient. Therefore, advantageously, at least a portion of the sample surface may include a surface treatment, such as a coating, to cause the adhered sample to adhere more strongly to the sample surface than to the walls. For example, the sample surface may include a gold capping layer to which a polymer is covalently bonded via thiol groups. The polymer may be a polymer containing positively charged groups. The polymer may be polyornithine or poly-d-lysine. The polymer may be a polymer electrolyte.

[0072] Alternatively or additionally, at least a portion of the wall surface may include a surface treatment, such as a coating, that causes the adhesive sample to adhere more weakly to the wall than to the sample surface. For example, the wall or a portion of the wall may be coated with a nonionic surfactant. The wall or a portion of the wall may be coated with Pluronic F-127, Poly-HEMA, or other poloxamers. If the wall comprises a photoresist material such as SU-8, the wall may be oxygen plasma treated and then coated with silane-terminated PEG molecules, such that the silane covalently bonds to the O2 plasma-treated surface and the PEG molecules form an anti-adhesion coating.

[0073] To achieve the desired geometric effect of the wall, at least a portion of the wall may have a height relative to the sample surface of more than 1 micrometer and less than 50 micrometers, preferably more than 2 micrometers and less than 20 micrometers, or more than 3 micrometers and less than 15 micrometers, and at least a portion of the wall may have a lateral thickness or maximum lateral thickness of more than 1 micrometer and less than 50 micrometers, preferably more than 5 micrometers and less than 40 micrometers.

[0074] To modify the effect of the wall to restrain or contain the adherent sample, at least a portion of the wall may have a rectangular cross-section, or a flared or rounded cross-section. For example, the cross-section of at least a portion of the wall may be stepped, or the thickness of the wall may vary non-monotonically. In particular, the cross-section of the wall may be flared so that the cross-section of the wall away from the sample surface is larger than the cross-section of the wall closest to the sample surface. For example, a portion of the wall may overhang the sample surface. This can improve containment of the adherent sample. Alternatively, the cross-section of the wall may be flared so that the cross-section of the wall away from the sample surface is smaller than the cross-section of the wall closest to the sample surface. This allows adherent cells to more easily reach the sample surface of the base. The wall preferably has the same cross-section throughout its length.

[0075] Flared walls, where the top is thicker than the base of the wall, or walls that overhang the sample surface, can advantageously provide a mechanical barrier to cell migration away from the sample surface. Alternatively, flared walls, where the top is less thick than the base of the wall, can advantageously allow any cells that may adhere to the wall to migrate to the sample surface. Thus, wall shapes may be optimized for different cell or sample types, as well as different wall and sample surface coatings.

[0076] Advantageously, the carrier may have lateral dimensions, preferably a minimum lateral dimension, greater than 5 micrometers and less than 300 micrometers, or preferably greater than 10 micrometers and less than 200 micrometers.

[0077] The walls of the carrier may be manufactured by any suitable method, but advantageously, for convenience and efficiency, the walls may comprise material that is also present in the base portion, and the walls are preferably manufactured using a process that is also used to manufacture the base portion. For example, the walls and base portion may conveniently be manufactured using a lithographic process, in which case magnetic material may also be incorporated into the carrier.

[0078] The walls may be fabricated using nanoimprint lithography or microimprint lithography.

[0079] In one embodiment, particularly if the carrier is fabricated using photolithography, the walls may comprise a structural photopolymer, preferably photoresist, SU-8 or AZ-10nXT. Alternatively or additionally, the walls may comprise a polymer such as a thermoplastic polymer, preferably PDMS.

[0080] In a further aspect, the present invention may advantageously provide a carrier system for an assay, comprising a plurality of carriers. In the carrier system, the carriers are preferably provided fixed to a substrate by a release layer as described above. The surface of the substrate on which the carriers are fixed is preferably flat.

[0081] In yet another aspect, the present invention advantageously provides a method for performing an assay using the carrier or carrier system described above. The method may advantageously include the steps of providing a plurality of carriers arranged on a substrate, introducing a biocompatible aqueous solution carrying the adhered sample to the carrier and the substrate such that the adhered sample adheres to the sample surface of the carrier, and peeling the carrier from the substrate. The carrier may be held in place on the substrate by a release layer between the carrier and the substrate, which release layer is activated by the biocompatible aqueous solution to peel the carrier. Additionally or alternatively, the carrier may be magnetic or include a magnetic material and may then be held in place on the substrate by an applied magnetic field and peeled from the substrate by changing or removing the applied magnetic field.

[0082] In a further aspect of the present invention, the carrier of the present invention is preferably magnetically operable, for example, during the assay stage. Thus, after peeling from the substrate, the solution during the assay can contain multiple substrates and carry adhesive samples. While in the solution, the carriers may move closer to each other, and it is important to avoid the adhesive sample from transferring from one carrier to another. The geometric effect of the walls surrounding the adhesive sample on each carrier can advantageously help keep the sample surfaces of different carriers spaced apart from each other when the carriers are in the solution, thus reducing the risk of the adhesive sample transferring from one carrier to another.

[0083] In some cases, individual carriers may be marked with a readable code, such as a barcode, or otherwise marked so that individual carriers or groups of carriers can be identified in the assay. When adherent samples are placed on individual carriers at the start of the assay, it is essential that the samples do not migrate from one carrier to another. Walls surrounding samples on carriers in solution can advantageously prevent sample migration between carriers, as they may be close to or pass through other carriers.

[0084] The carrier may include a readable code applied to the sample surface of the carrier. Alternatively, the readable code may comprise one or more holes or one or more areas of different material defined through or in the base of the carrier. Preferably, the wall includes the readable code. Advantageously, a readable code included in the wall prevents the code from reducing the area of ​​the sample surface available for cell attachment. The code may comprise a plurality of dots, dashes, or dots and dashes. The code may include several lines. The code may partially penetrate the wall. The code may penetrate through the wall to the carrier base. The code may be formed by lithography.

[0085] The readable code can take various forms as a marking in the carrier that encodes information that identifies the carrier on which it is applied. When imaged, the pattern on the base or wall of a given carrier can be analyzed for its coded information and therefore used to identify that particular carrier. The code is preferably readable by image analysis of a microscopic image.

[0086] Also, in an assay, magnetically manipulable carriers may generally be manipulated onto a substrate, e.g., for imaging or analysis. When carriers are positioned on a substrate in this manner, walls can advantageously prevent the transfer of adhered samples from the carrier to the substrate or from one carrier to another. In addition, as noted above, carriers moving through a solution during an assay may be in close proximity to or even in contact with other carriers, and walls can advantageously prevent the transfer of samples between such carriers. These effects can only be achieved by placing walls on the carrier itself. While prior art has used walls to position or restrain adhered samples onto a substrate, the walls of the prior art are fixed to the substrate and therefore have no effect after carriers, such as magnetically manipulable carriers, are moved or manipulated toward a new substrate during an assay.

[0087] Additionally, when the carrier is driven through the assay medium by an external magnetic field, the assay sample carried by the carrier is subjected to shear forces. These forces can be harmful to the assay sample, and the presence of a wall adjacent to the assay sample on the sample face of the carrier can advantageously reduce the shear forces experienced by the assay sample.

[0088] The present invention is defined in the claims. However, below is provided a non-exhaustive list of non-limiting examples. Any one or more of the features in these examples may be combined with any one or more features in another example, embodiment, or aspect described herein.

[0089] (Example)

[0090] [Example 1] A carrier for an adhesion assay sample, comprising a sample surface for receiving the adhesion assay sample and a peripheral region surrounding the sample surface and positioned between the sample surface and an edge of the carrier, the carrier comprising a surface treatment such that the adhesion assay sample adheres more weakly to the peripheral region than to the sample surface.

[0091] [Example 2] A carrier as described in Example 1, wherein, when in use, the carrier is removably positionable on a substrate for receiving the adhesion assay sample, and when the carrier is positioned on the substrate, a peripheral region is between the sample surface and the substrate, and preferably surrounds the sample surface.

[0092] [Example 3] A carrier as described in Example 1 or 2, wherein when the carrier is placed on the substrate, the height of the sample surface and the surrounding area relative to the substrate differs by less than 10% of the maximum thickness of the carrier, preferably by less than 1% or 0.1% of the maximum thickness of the carrier.

[0093] [Example 4] A carrier according to any one of Examples 1 to 3, wherein the thickness of the carrier in the sample surface region differs from the thickness of the carrier in the peripheral region by less than 10%, preferably less than 1% or 0.1%.

[0094] [Example 5] 5. The carrier according to any one of Examples 1 to 4, wherein the peripheral region is substantially flat and preferably parallel to the substrate when the carrier is placed on the substrate.

[0095] [Example 6] 6. The carrier of any one of Examples 1 to 5, wherein the sample surface is substantially flat and preferably parallel to the substrate when the carrier is placed on the substrate.

[0096] [Example 7] The carrier according to any one of Examples 1 to 6, wherein the carrier comprises a base portion, and an upper surface of the base portion forms the sample surface and the peripheral region.

[0097] [Example 8] 8. The carrier of any one of Examples 1 to 7, wherein the peripheral region comprises a sample adhesion reducing coating.

[0098] [Example 9] 9. The carrier of Example 8, wherein the coating on the peripheral region comprises a non-ionic surfactant.

[0099] [Example 10] 10. The carrier of any one of Examples 8 and 9, wherein the coating on the peripheral region comprises one or more selected from Pluronic F-127, Poly-HEMA, PEG polymer, and poloxamer.

[0100] [Example 11] 11. The carrier of any one of Examples 1 to 10, wherein the sample surface comprises a sample adhesion promoting coating.

[0101] [Example 12] 12. The carrier of Example 11, wherein the sample surface comprises a biofunctionalized coating adapted to receive the adhesion assay sample.

[0102] [Example 13] 13. The carrier of claim 11 or 12, wherein the sample surface comprises a charged polymer.

[0103] [Example 14] A carrier described in any one of Examples 1 to 13, wherein at least a portion of the peripheral region has a lateral width or maximum lateral thickness greater than 1 micrometer and less than 50 micrometers, or greater than 5 micrometers and less than 40 micrometers.

[0104] [Example 15] 15. A carrier according to any one of Examples 1 to 14, wherein the carrier has lateral dimensions, preferably smallest lateral dimensions, greater than 5 micrometers and less than 300 micrometers, or preferably greater than 10 micrometers and less than 200 micrometers.

[0105] [Example 16] The carrier according to any one of Examples 1 to 15, wherein the carrier comprises a magnetic material.

[0106] [Example 17] A carrier system for assays, comprising a plurality of carriers according to any one of Examples 1 to 16.

[0107] [Example 18] A method for performing an assay using a carrier according to any one of Examples 1 to 16 or a carrier system according to Example 17, comprising the steps of: providing a plurality of substrates arranged on a substrate; introducing a biocompatible aqueous solution carrying the adhesion assay sample to the carrier and the substrates so that the adhesion assay sample adheres to the sample surface of the carrier but not to the surrounding area; and peeling the carrier from the substrate.

[0108] [Example 19] 19. A method of performing the assay of Example 18, wherein the carrier is held in place on the substrate by a release layer between the carrier and the substrate, the release layer being activated by the biocompatible aqueous solution to release the carrier.

[0109] [Example 20] 20. A method of performing an assay as described in Example 18 or 19, wherein the carrier is magnetic or comprises a magnetic material, is held in place on a substrate by an applied magnetic field, and is detached from the substrate by changing or removing the applied magnetic field.

[0110] [Example 21] A carrier for an adhesion assay sample, the carrier comprising, in use, a sample surface for receiving the adhesion assay sample, and a wall between the sample surface and an edge of the carrier.

[0111] [Example 22] 22. The carrier of Example 21, wherein, in use, the adhesion assay sample adheres more strongly to the sample surface than to the wall.

[0112] [Example 23] A carrier as described in Example 21 or 22, wherein, in use, the carrier is removably positionable on a substrate for receiving the adhesion assay sample, and when the carrier is positioned on the substrate, the wall is between the sample surface and the substrate.

[0113] [Example 24] 24. The carrier of any one of Examples 21 to 23, wherein the wall surrounds the sample surface.

[0114] [Example 25] A carrier according to any one of Examples 21 to 24, wherein the sample surface is substantially flat and preferably parallel to the substrate when the carrier is placed over the substrate.

[0115] [Example 26] A carrier described in any of Examples 21 to 25, wherein the carrier comprises a base portion, an upper surface of the base portion forms the sample surface, and the wall extends above the upper surface of the base portion so as to be adjacent to or spaced from the sample surface.

[0116] [Example 27] A carrier according to any one of Examples 21 to 26, wherein at least a portion of the sample surface comprises a coating that causes the adhesion assay sample to adhere more strongly to the sample surface than to the wall.

[0117] [Example 28] A carrier according to any one of Examples 21 to 27, wherein at least a portion of the surface of the wall comprises a coating such that the adhesion assay sample adheres less strongly to the wall than to the sample surface.

[0118] [Example 29] The carrier of any one of Examples 21 to 28, wherein at least a portion of the wall has a height relative to the sample surface that is greater than 1 micrometer and less than 25 micrometers.

[0119] [Example 30] A carrier according to any one of Examples 21 to 29, wherein at least a portion of the wall has a lateral thickness, or a maximum lateral thickness, greater than 1 micrometer and less than 40 micrometers.

[0120] [Example 31] A carrier according to any one of Examples 21 to 30, wherein at least a portion of the wall has a rectangular cross-section, or a flared or rounded cross-section.

[0121] [Example 32] A carrier according to any one of Examples 21 to 31, having lateral dimensions, preferably smallest lateral dimensions, greater than 5 micrometers and less than 300 micrometers, or preferably greater than 10 micrometers and less than 200 micrometers.

[0122] [Example 33] The carrier according to any one of Examples 21 to 32, wherein the carrier comprises a magnetic material.

[0123] [Example 34] A carrier described in any of Examples 21 to 33, wherein the carrier comprises a base portion, the upper surface of the base portion forms the sample surface, and the wall comprises a material that is also present in the base portion, preferably manufactured using a process that is also used to manufacture the base portion.

[0124] [Example 35] A carrier described in any of Examples 21 to 34, wherein the carrier comprises a base portion, an upper surface of the base portion forms the sample surface, and the wall and the base portion are manufactured using a lithography process.

[0125] [Example 36] The carrier according to any of Examples 21 to 35, wherein the wall comprises a structural photopolymer, preferably photoresist, SU-8 or AZ-10nXT.

[0126] [Example 37] The carrier of any of Examples 21-36, wherein the wall comprises a thermoplastic polymer, preferably a polymer such as PDMS.

[0127] [Example 38] The carrier of any one of Examples 21 to 37, wherein the sample surface comprises a gold capping layer, and a polymer is covalently bonded to the gold capping layer via a thiol group.

[0128] [Example 39] A carrier system for assays, comprising a plurality of carriers according to any one of Examples 21 to 38.

[0129] [Example 40] A method for performing an assay using a carrier described in any one of Examples 21 to 38 or a carrier system described in Example 39, comprising the steps of providing a plurality of carriers arranged on a substrate; introducing a biocompatible aqueous solution carrying an adhesion assay sample to the carriers and the substrate so that the adhesion assay sample adheres to the sample surface of the carrier; and peeling the carrier from the substrate.

[0130] [Example 41] A method of performing the assay of Example 40, wherein the carrier is held in place on the substrate by a release layer between the carrier and the substrate, and the release layer is activated by a biocompatible aqueous solution to release the carrier.

[0131] [Example 42] 42. A method for performing an assay according to Example 40 or 41, wherein the carrier is magnetic or comprises a magnetic material, is held in place on a substrate by an applied magnetic field, and is detached from the substrate by changing or removing the applied magnetic field.

[0132] Specific embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0133] [Figure 1] 1 is a schematic cross-sectional view of three carriers fixed to a substrate according to a first embodiment of the present invention; [Figure 2] 2 is a schematic cross-sectional view of the carrier of FIG. 1 after peeling from the substrate. [Figure 3]3 is a schematic three-quarter view of the carrier of FIGS. 1 and 2. [Figure 4] 1 is a photomicrograph of an array of carriers embodying the present invention fixed to a substrate. [Figure 5] 1 is a photomicrograph of a number of carriers embodying the present invention carrying adherent cells and suspended in an assay solution. [Figure 6] 1 is a schematic cross-sectional view, with the vertical dimension exaggerated for clarity, of three carriers according to a second embodiment of the invention attached to a substrate by a release layer; [Figure 7] 7 is a schematic cross-sectional view of the carrier of FIG. 6 after peeling from the substrate. [Figure 8] 7A-7C show schematic cross-sectional views of five alternative embodiments of the invention similar to the carrier of FIG. 6 after peeling from the substrate, with different cross-sectional shapes of the carrier walls. [Figure 9] FIG. 8 is a plan view of a micrograph of a substrate supporting an array of carriers of FIGS. 6 and 7. [Figure 10] 10 is a micrograph of a carrier in a second embodiment, carrying adherent cells and suspended in an assay solution. DETAILED DESCRIPTION OF THE INVENTION

[0134] 1, 2, and 3 show a carrier according to a first embodiment of the present invention. As shown in FIG. 1, a carrier 100 is first provided for the assay, immobilized on a substrate 12 in the form of a silicon chip by a release layer 14. The release layer 14 may comprise dextran. The substrate is sized to support a plurality of carriers, perhaps 1,000 to 500,000, depending on the application of the assay. Each carrier is suitable for receiving one or more adherent cells.

[0135] The (or each) carrier 100 is fabricated as follows: A release layer 14 is spin-coated onto a silicon substrate 12. A square layer 16 of structural photopolymer is then deposited on the release layer. The shape of the photopolymer is defined by photolithography and is approximately 1.5 micrometers thick with lateral dimensions of 120 micrometers along each side of the square. The shape of the photopolymer is therefore a flat, i.e., high aspect ratio, rectangular parallelepiped with relatively large square top and bottom surfaces. The photopolymer may be SU-8. The square layer of photopolymer forms the base or body of the carrier.

[0136] A multi-layer magnetic heterostructure 20 is deposited in the central square portion of the top surface of the photopolymer layer 16 using lithographic techniques such as those described in US Pat. No. 6,229,999. To define the shape of the sample surface, the magnetic heterostructure is deposited only in the central portion of the photopolymer layer.

[0137] A functionalizable gold cap 22 is then deposited on the multilayer magnetic heterostructure 20, followed by a layer 24 of polymer to which a target adhesion sample can readily adhere. This polymer layer 24 defines the sample face 25 of the carrier for receiving the assay sample when the assay is performed. The sample face is centrally located on the carrier base and is square in shape with sides of approximately 100 micrometers.

[0138] In this embodiment, the peripheral region 28 surrounding the sample surface is 10 micrometers wider.

[0139] The polymer of the polymer layer 24 contains thiol groups and is applied by immersing the carrier and substrate in a solution containing the polymer. The polymer is covalently bonded to the gold caps 22 of the carrier through the thiol groups. The solution contains a non-aqueous solvent, such as ethanol. The release layer is insoluble in such non-aqueous solvents, so the carrier remains fixed to the substrate while immersed in the solution containing the polymer.

[0140] The cumulative thickness of the magnetic heterostructure, gold cap, and polymer layer is less than 1 micrometer, and typically a few hundred nanometers, depending on the thickness of the deposited polymer layer in particular, so that the top surface of the finished carrier is nearly flat, or planar.

[0141] Each carrier may include a readable code, such as a barcode. A predetermined code is assigned to each carrier to identify it. For example, the predetermined code may indicate the specific adherent cells received on the carrier, or the specific adherent cells and the specific reagents to which the adherent cells have been exposed as part of an assay. In other words, the code allows for multi-channel assays to be performed using multiple carriers. The code may be defined in a peripheral region of the carrier. In one example, the code includes multiple dots or dashes defined in the peripheral region by photolithography, for example, by incorporating holes or depressions in an SU-8-based layer in a single exposure.

[0142] Carriers embodying the present invention may also be realized by forming a magnetic heterostructure throughout the carrier and then defining the area and shape of the sample surface 25 as part of the top surface of the magnetic heterostructure. This can be done by depositing a gold cap or polymer coating on selected areas of the carrier surface, which are surrounded by a peripheral region 28 that extends around the edges of the carrier surface. Thus, the sample surface occupies a relatively central location on the carrier surface, and the peripheral region 28 extends around the entire periphery of the sample surface, encompassing the area between the sample surface and the carrier edge.

[0143] In a more preferred embodiment, the sample surface 25 is about 100 micrometers square and the width of the peripheral region is about 20 micrometers between the edge of the carrier and the edge of the sample surface.

[0144] The sample surface 25 and the peripheral region 28 may have different coatings applied to them, may be of different materials, or may have undergone different material treatments so that the assay sample adheres more strongly to the sample surface 25 than to the peripheral region 28.

[0145] Thus, for example, after forming sample surface 25 as described above, a peripheral region 28 on the top surface of photopolymer layer 16 is coated with an adhesion-reducing coating material to reduce or prevent adhesion of the assay sample (e.g., sample cells) to peripheral region 28 of carrier 10. The peripheral region coating can be formed by depositing a non-ionic surfactant, such as Pluronic F-127, in a strip surrounding the central sample surface. The sample surface can also be coated with, for example, a gold cap without the low-adhesion surface treatment.

[0146] In a different embodiment, instead of lithographically depositing the gold cap 22 only on the central sample surface 25, the gold cap may be formed over the entire top surface of the carrier 100, and each coating or coatings that define the sample surface may be applied only to a defined area on the gold cap. For example, the gold cap may be functionalized only at the center of the carrier of the sample surface, while an adhesion-reducing coating may be applied to the exposed portion of the gold cap at the periphery of the carrier to surround the sample surface.

[0147] In other embodiments, a coating other than the polymer coating 24 described above may be used to coat the sample surface. For example, an adhesion-promoting sample surface coating can have multiple ligands, including antibodies that specifically bind to cell receptors such as integrins, extracellular matrix proteins such as collagen, or Matrigel, a protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Instead of applying a coating to the sample surface, the method can include making the physical surface of the sample surface particularly suitable for receiving adherent cells, for example, by modifying a gold cap.

[0148] In other embodiments, various adhesion-reducing coatings may be used to reduce adhesion of the sample to the peripheral regions 18, 28. For example, the peripheral regions may be coated with Poly-HEMA or another poloxamer.

[0149] Figure 4 is a plan view micrograph of an array of carriers immobilized on a substrate. Any number of carriers can be immobilized on a substrate as required for any assay application.

[0150] The final step in the process of fabricating the carrier system is sterilization, which is not shown in the diagram. To sterilize the carrier system, the array of substrate-mounted carriers is placed in a sterile package, sealed, and then placed in an oven at 120 °C for 4 hours. This sterilizes the carrier system and creates a ready-to-use sample that can be opened in a sterile cell culture environment.

[0151] 5 is a photomicrograph of a plurality of carriers embodying the present invention suspended in an assay solution in use in an adherent cell assay. In the photomicrograph, the magnetic carriers are aligned to face the camera by proper orientation of an external magnetic field, and the cells have been stained to improve their visibility. Cells can be seen adhering to the sample surface of each carrier and not to the surrounding area surrounding the sample surface of each carrier.

[0152] To place cells on the sample surface of the carrier in Figure 5, the following steps were performed. A carrier system, such as an array of carriers on a silicon substrate, is placed in a container and immersed in a biocompatible aqueous solution containing adherent cells. When the biocompatible aqueous solution comes into contact with the release layer 14, the release layer 14 dissolves and detaches the carrier. If additional time is needed for the cells to adhere to the carrier while it is on the substrate, an external magnetic field is applied to keep the carrier in contact with the substrate even after the release layer detaches the carrier.

[0153] Once the cells adhere to the carriers, they are detached from the substrate and move freely in the aqueous solution surrounding the carrier system. An external magnetic field can be used to apply a force to the carriers to manipulate and move them as desired to perform assays. As shown in Figure 5, a magnetic field can be used to align the carriers for photographic imaging.

[0154] The structure of a carrier 210 according to a second embodiment of the invention is shown in Figures 6 and 7. Figure 6 shows three carriers in the form of silicon chips fixed to a substrate 212 by a release layer 214. The substrate is sized to receive a number of carriers, perhaps 1000 to 500,000, depending on the assay application. Each carrier is suitable for receiving one or more adherent cells.

[0155] The (or each) carrier 210 is fabricated as follows: A release layer 214 is spin-coated onto a silicon substrate 212. The release layer 214 may include dextran. A square layer 216 of structural photopolymer is then deposited onto the release layer. The shape of the photopolymer is defined by photolithography and is approximately 1.5 micrometers thick with lateral dimensions of 160 micrometers along each side of the square. The shape of the photopolymer is therefore a flat, i.e., high-aspect-ratio, rectangular parallelepiped with relatively large square top and bottom surfaces. The photopolymer may include SU-8. Upstanding walls 218 of photopolymer are then formed along the edges of the square layer 216, again by photolithography. The walls are 7 micrometers high and 20 micrometers thick.

[0156] At least a portion of the wall is then subjected to a surface treatment that reduces adhesion of the adhesive sample to the wall. The coating can be performed, for example, by immersion in an ethanol solution containing Pluronic F127, which bonds to the hydrophobic photopolymer comprising the wall. Alternatively, the wall surface can be treated with oxygen plasma and then immersed in a silane-PEG ethanol solution, in which the silane-PEG covalently bonds to the oxygen plasma-activated surface. (In contrast, in a preferred embodiment, the sample surface can include a gold layer, which may differ in its hydrophobicity from the wall photopolymer and does not form surface -OH groups during plasma treatment. Therefore, a low-adhesion surface treatment may not be performed on the sample surface.)

[0157] In some embodiments, it may be desirable to apply the low-adhesion surface treatment to only certain portions of the wall. This can be achieved by masking. For example, certain areas of the wall can be prevented from being coated by patterning a sacrificial mask of photoresist before applying the low-adhesion surface treatment.

[0158] Optionally, in some embodiments, it may be desirable to additionally apply a surface treatment to the substrate between the carriers to reduce any adhesion of the sample to the substrate. This may only be necessary for certain applications of the carrier system where adhesion of the sample to the substrate may be an issue. The exposed release layer on the substrate dissolves during use when the carrier system is exposed to a biocompatible aqueous solution carrying the adhered sample; therefore, to apply the surface treatment between the carriers, the exposed release layer may be removed from the substrate, and a surface treatment such as Pluronic 127 or silane-PEG may be applied to the substrate between the carriers.

[0159] On top of the layer of photopolymer 216, within the walls 218, a multilayer magnetic heterostructure 220 is deposited (as described in US Pat. No. 6,213,999).

[0160] A functionalizable gold cap 222 is then deposited on the multilayer magnetic heterostructure 220, followed by a layer of polymer 224 to which a target adhesion sample can readily adhere. This polymer layer 224 defines the sample face of the carrier for receiving the assay sample when the assay is performed, and the sample face is approximately 100 micrometers square.

[0161] The polymer of the polymer layer 224 contains thiol groups and is applied by immersing the carrier and substrate in a solution containing the polymer. The polymer is covalently bonded to the gold caps 222 of the carrier through the thiol groups. The solution contains a non-aqueous solvent, such as ethanol. The release layer is insoluble in such non-aqueous solvents, and therefore the carrier remains fixed to the substrate while immersed in the solution containing the polymer.

[0162] The cumulative thickness of the magnetic heterostructure, gold cap, and polymer layer is less than 1 micrometer, and is typically a few hundred nanometers, depending especially on the thickness of the deposited polymer layer, resulting in walls extending approximately 6-7 micrometers above the sample surface.

[0163] Each carrier may include a readable code, such as a barcode. To identify the carrier, each carrier is assigned a predetermined readable code. For example, the predetermined barcode can then identify the carrier carrying specific adherent cells, as well as any specific reagents to which the adherent cells have been exposed as part of the assay. In other words, the code allows for multi-channel assays to be performed with multiple carriers. The code may be defined on the walls of the carrier. In one embodiment, for example, a barcode of dots and dashes or lines can be included in the lithographic pattern on the wall. This can be as simple as multiple dots, dashes, or lines, or as complex as an actual barcode. When imaged, the pattern on the wall of a given carrier can be analyzed for its barcoding information.

[0164] As noted above, in other embodiments, different wall shapes or cross sections may be appropriate for different cell or sample types, and wall and sample surface coatings. Some examples are shown in FIG.

[0165] The first two examples in Figure 8 (reading from left to right) show flared walls where the top is thicker than the base of the wall, resulting in the wall overhanging the sample surface. At the same time, the outer surface of the wall may be vertical, perpendicular to the base of the carrier, or flared outward. Advantageously, such walls can provide a mechanical barrier to cell migration away from the sample surface and can limit cell migration toward the substrate between carriers.

[0166] Alternatively, flared walls may be used, in which the top of the wall is thinner than the base of the wall, as shown in the top and bottom right of Figure 8. Advantageously, such a wall shape can encourage any cells that may adhere to the wall to migrate to the sample surface.

[0167] In these variations of the wall shape, the wall cross section may be stepped, as shown in the lower left of Figure 8. A stepped wall shape may be easier to manufacture than a continuous flared wall while retaining the functional advantages of a flared wall.

[0168] The walls may combine features of these wall shapes, for example, the walls may have an inwardly flared inner surface (overhanging the sample surface) and an inwardly flared outer surface (sloping away from the surrounding substrate), or vice versa.

[0169] Thus, the wall geometry can be optimized for different cell or sample types and different wall and sample surface treatments.

[0170] 9 is a plan view of an array of carriers according to the second embodiment fixed to a substrate. Any number of carriers can be fixed to the substrate depending on the assay application.

[0171] In other embodiments, surface treatments other than the polymer coatings described above for the sample face can be used. For example, the coating can have multiple ligands, including antibodies that specifically bind to cell receptors such as integrins, or extracellular matrix proteins such as collagen, or Matrigel, a protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Instead of applying a coating, the method can include modifying the physical surface of the sample face of the carrier to make it particularly suitable for receiving adherent cells, for example, by modifying a gold cap.

[0172] The final step in the process of manufacturing the carrier system is sterilization, which is not shown in the diagram. To sterilize the carrier system, the carrier system is placed in sterile packaging, sealed, and then placed in a 120°C oven for 4 hours. This sterilizes the carrier system and creates a ready-to-use sample that can be opened in a sterile cell culture environment.

[0173] 10 is a micrograph of a plurality of carriers of the second embodiment suspended in an assay solution during use in an assay of adherent cells. In the micrograph, the magnetic carriers are aligned to face the camera by proper orientation of the external magnetic field, and the cells are stained to improve their visibility. It can be seen that the cells adhere to the sample surface of each carrier, but not to the walls surrounding the sample surface of each carrier.

[0174] To place cells on the sample surface of the carrier in FIG. 10, the following steps were performed: A carrier system, such as the array of carriers on a silicon substrate shown in FIG. 9, is placed in a container and immersed in a biocompatible aqueous solution containing adherent cells. When the biocompatible aqueous solution comes into contact with the release layer 214, the release layer 214 dissolves and detaches the carrier. If additional time is needed to allow the cells to adhere to the carrier while the carrier is on the substrate, an external magnetic field is applied to keep the carrier in contact with the substrate even after the release layer detaches the carrier.

[0175] Once the cells adhere to the carriers, they are detached from the substrate and move freely in the aqueous solution surrounding the carrier system. An external magnetic field can be used to apply a force to the carriers to manipulate and move them as desired to perform assays. As shown in Figure 10, a magnetic field can be used to align the carriers for photographic imaging.

Claims

1. A carrier for an adhesion assay sample, comprising: a sample surface for receiving the adhesion assay sample; and a peripheral region surrounding the sample surface and positioned between the sample surface and an edge of the carrier, the peripheral region comprising a sample adhesion reducing coating such that, in use, the adhesion assay sample adheres more weakly to the peripheral region than to the sample surface.

2. 2. A carrier as described in claim 1, wherein, when in use, the carrier is removably positionable on a substrate for receiving the adhesion assay sample, and when the carrier is positioned on the substrate, the peripheral region is between the sample surface and the substrate, and preferably surrounds the sample surface.

3. 3. The carrier of claim 2, wherein when the carrier is placed on the substrate, the height of the sample surface and the peripheral region relative to the substrate differs by less than 10% of the maximum thickness of the carrier, preferably by less than 1% or 0.1% of the maximum thickness of the carrier.

4. A carrier according to any one of claims 1 to 3, wherein the thickness of the carrier in the region of the sample surface and the thickness of the carrier in the peripheral region differ by less than 10%, preferably less than 1% or 0.1%.

5. A carrier according to any one of claims 1 to 4, wherein the peripheral region is substantially flat, preferably parallel to the substrate when the carrier is placed on the substrate.

6. 6. A carrier according to any one of claims 1 to 5, wherein the sample surface is substantially flat, preferably parallel to the substrate when the carrier is placed on the substrate.

7. 7. A carrier according to any one of claims 1 to 6, wherein the carrier comprises a base portion, the upper surface of the base portion forming the sample surface and the peripheral region.

8. A carrier according to any one of claims 1 to 7, comprising a wall between the sample surface and an edge of the carrier.

9. The carrier of claim 8 , wherein the peripheral region comprises the wall.

10. 10. A carrier as described in claim 8 or 9, wherein at least a portion of the surface of the wall comprises the sample adhesion reducing coating such that, in use, the adhesion assay sample adheres more weakly to the wall than to the sample surface.

11. A carrier according to any one of claims 8 to 10, wherein the wall surrounds the sample surface.

12. A carrier according to any one of claims 8 to 11, wherein when the carrier is placed on the substrate, the wall is between the sample surface and the substrate.

13. 13. A carrier according to any one of claims 8 to 12, wherein the carrier comprises a base portion, an upper surface of the base portion forms the sample surface, and the wall extends above the upper surface of the base portion so as to be adjacent to or spaced from the sample surface.

14. 14. The carrier according to claim 8, wherein at least a portion of the wall has a height relative to the sample surface that is greater than 1 micrometer and less than 25 micrometers.

15. A carrier according to any one of claims 8 to 14, wherein at least a part of the wall has a lateral thickness, or a maximum lateral thickness, of more than 1 micrometer and less than 40 micrometers.

16. A carrier according to any one of claims 8 to 15, wherein at least a part of the wall has a rectangular cross section, or a flared or rounded cross section.

17. 17. A carrier according to any one of claims 8 to 16, wherein the carrier comprises a base portion, the upper surface of the base portion forming the sample surface, and the wall comprises a material that is also present in the base portion, preferably manufactured using a process that is used to manufacture the base portion.

18. 18. A carrier according to any one of claims 8 to 17, wherein the carrier comprises a base portion, an upper surface of the base portion forms the sample surface, and the walls and the base portion are manufactured using a lithographic process.

19. A carrier according to any one of claims 8 to 18, wherein the walls comprise a structural photopolymer, preferably photoresist, SU-8 or AZ-10nXT.

20. A carrier according to any one of claims 8 to 19, wherein the wall comprises a polymer such as a thermoplastic polymer, preferably PDMS.

21. A carrier according to any one of claims 1 to 20, wherein the coating of the peripheral region comprises a non-ionic surfactant.

22. 22. The carrier according to any one of claims 1 to 21, wherein the coating of the peripheral region comprises one or more selected from Pluronic F-127, Poly-HEMA, PEG polymers and poloxamers.

23. 23. A carrier according to any preceding claim, wherein the sample surface comprises a sample adhesion promoting coating.

24. 24. The carrier of claim 23, wherein the sample surface comprises a bio-functionalized coating adapted to receive the adhesion assay sample.

25. 25. A carrier according to claim 23 or 24, wherein the sample surface comprises a charged polymer.

26. 26. A carrier according to any one of claims 1 to 25, wherein at least a portion of the peripheral region has a lateral width or a maximum lateral thickness greater than 1 micrometer and less than 50 micrometers, or greater than 5 micrometers and less than 40 micrometers.

27. 27. A carrier according to any one of the preceding claims, wherein the carrier has lateral dimensions, preferably smallest lateral dimensions, greater than 5 micrometers and less than 300 micrometers, preferably greater than 10 micrometers and less than 200 micrometers.

28. 28. The carrier according to any one of claims 1 to 27, wherein the carrier comprises a magnetic material.

29. A carrier system for an assay, comprising a plurality of carriers according to any one of claims 1 to 28.

30. A method for performing an assay using a carrier according to any one of claims 1 to 28 or using a carrier system according to claim 29, comprising the steps of: providing a plurality of carriers arranged on a substrate; introducing a biocompatible aqueous solution carrying the adhesion assay sample to the carriers and the substrate so that the adhesion assay sample adheres to the sample surface of the carrier but not to the surrounding area; and peeling the carrier from the substrate.

31. 31. A method of performing the assay of claim 30, wherein the carrier is held in place on the substrate by a release layer between the carrier and the substrate, the release layer being activated by the biocompatible aqueous solution to release the carrier.

32. 32. A method of performing an assay according to claim 30 or 31, wherein the carrier is magnetic or comprises a magnetic material and is held in place on a substrate by an applied magnetic field and is detached from the substrate by changing or removing the applied magnetic field.

Images