Carrier, carrier system and method

Abstract

A carrier (100) for adherent detection of a sample is provided. The carrier includes a sample surface (25) for receiving an adherent sample and a peripheral region (28) between the sample surface and an edge of the carrier. The peripheral region includes a coating with reduced sample adhesion such that, in use, the adherent sample adheres to the peripheral region weaker than to the sample surface.

Description

[0001] The present invention relates to a carrier, a carrier system and a method, and in particular to a carrier system for detection and a method for manufacturing the carrier system. Background Art

[0002] An assay is a research procedure used to qualitatively or quantitatively measure the presence, amount, or activity of an analyte in a test sample. Typically, the test sample is a cell or cell culture, and the analyte is a protein or gene sequence from that cell or cell culture, but analytes more broadly can include metabolites, peptides, proteins, nucleic acids, extracellular vesicles, organelles, cells, or tissues.

[0003] Some biological test samples (such as certain types of cells) can be suspended in aqueous solutions. However, there are many biological test samples that cannot be easily suspended in solution and are most active when attached to a surface (such as adherent cells). Testing of such samples, including the measurement step, usually needs to be performed when the test sample is attached to the bottom of the well plate. In such tests, the test process may be disadvantageously limited by the fact that the test sample attached to the bottom of the well plate cannot be easily transferred to another container without peeling it from the bottom of the well plate. This may damage the sample.

[0004] An improved approach to testing with adherent test samples (such as adherent cells) is to use carriers or particles to carry the test samples. Examples of such carrier systems are described in patent publications WO 2020 / 099846 and WO 2021 / 224631.

[0005] In such a system, multiple carriers secured to a substrate can be provided, and adherent cells can be transferred to the exposed surfaces of the carriers, for example, from a biocompatible solution. The carriers can be magnetic and then fixed to the substrate (via an applied magnetic field), allowing time for the sample to attach to the carriers. The magnetic carriers, along with the adherent sample, can then be released from the substrate and guided or manipulated by the applied magnetic field to complete the assay.

[0006] However, when adherent samples are received on a carrier, especially if multiple carriers are positioned close to each other on a substrate, there arises the problem that the sample may adhere to or migrate to the substrate, or the sample may adhere to both the substrate and the carrier.

[0007] Methods of spatially restricting cell growth are known for certain applications.

[0008] Cells are routinely grown or cultured on polydimethylsiloxane (PDMS) substrates (see "Large area micropatterning of cells on polydimethylsiloxane surfaces," Journal of Biological Engineering, Vol. 8, No. 24, 2014, by Moustafa et al.). This method does not involve the use of a carrier but instead describes a method for spatially controlling and patterning cell growth on PDMS substrates. In this method, photoactivated graft polymerization of polyethylene glycol diacrylate (PEG-DA) is used to form patterned cell adhesion barriers on the substrate. This encourages cell growth within defined microchannels between the barriers.

[0009] Another method is described by Wang et al. in "Micropallet arrays with poly(ethylene glycol) walls" in "The Royal Society of Chemistry's Lab Chip" 2008, Vol. 8, pp. 734-740. Wang et al. prepared a microwell array on a glass substrate, each microwell including a planar microstage of SU-8 at its bottom. Adherent cells can be received onto the microstage in the microwell, and then a single microstage can be driven out of its microwell by a focused laser pulse directed through the glass substrate below any desired microstage. The cells carried by the released microstage can then be collected, cultured, and cloned and expanded. Wang et al. described two methods for forming microwells. One is to form virtual air walls between the microstages by applying a hydrophobic coating to the glass substrate, and to form bubbles adhered to the substrate by Cassie-Baxter wetting. Another approach is to form photopolymerized PEG hydrogel walls on glass substrates, using a method similar to that of Moustafa et al., but by localizing the photopolymerization by defining the PEG walls between the microstages using the difference in UV transmittance between the glass substrate and the SU-8 microstage. Individual microstages can still be separated from between the walls on the substrate using focused laser pulses.

[0010] However, when using magnetically controllable carriers such as those described in WO2020 / 099846 and WO2021 / 224631, several problems arise if these methods are used to prevent cells from adhering to the substrate. First, the presence of a wall on the substrate between the carriers may hinder the release of the carrier from the substrate. Wang et al. used laser pulses to actively drive their microstage away from the substrate. But if the carrier or microstage is released from the substrate only by removing the applied magnetic field, they may be retained in place by the wall. Another problem is that the method of Wang et al. requires the use of a transparent glass substrate, which provides both a photomask to achieve the formation of the wall and the use of laser pulses to achieve the separation of the microstage.

[0011] Conventional use of carriers for adherent test samples raises further issues when magnetic carriers are utilized and an applied magnetic field is used to guide or manipulate the carrier through the test. In these cases, adherent test samples can be subjected to significant shear forces as they are driven through the test medium (typically a biocompatible solution). This is not a problem for many test samples, but for large samples with lower levels of adhesion, shear forces can be detrimental to test results.

[0012] The present invention aims to solve these problems. Summary of the Invention

[0013] The present invention advantageously provides a carrier for detecting samples adhered to a wall, a carrier system and a method as defined in the appended independent claims, which should now be incorporated by reference. Preferred or advantageous features of the invention are listed in the dependent claims.

[0014] In a first aspect, the present invention may therefore provide a carrier for adhering to a sample for detection. The carrier may comprise a sample surface for receiving the adhered sample and a peripheral region surrounding the sample surface and located between the sample surface and the edge of the carrier. At least part of the carrier comprises one or more coatings such that the adhered sample has a weaker adhesion to the peripheral region than to the sample surface. To achieve this, a suitable coating may be applied to the sample surface, or to the peripheral region, or to both the sample surface and the peripheral region. Preferably, the peripheral region comprises a coating that reduces sample adhesion. The presence of the coating(s) on the carrier may advantageously promote adhesion of the adhered sample to the sample surface, which forms a defined region spaced apart from or away from the edge of the carrier.

[0015] The coating(s) can advantageously ensure that, during use, adherent samples adhere more strongly to the sample surface than to surrounding areas. This can advantageously mean that the sample adheres only to a limited area of ​​the carrier surface, which can make analysis of the carrier to which the sample adheres easier. For example, analyzing a sample adhered to the central sample surface of the carrier, which is more easily viewed from above, may be simpler than analyzing a sample adhered to the periphery or edge of the carrier.

[0016] The surface of the carrier, including the sample surface and the surrounding area, is preferably flat, planar, or substantially flat or planar. For example, the body or base of the carrier may include a flat or planar surface for supporting the sample, which surface may then be treated or coated to enhance or promote adhesion of the sample to the sample surface and / or to reduce or inhibit adhesion of the sample to the surrounding area.

[0017] The surface treatment (such as a coating) applied to the sample surface and / or the surrounding area may have varying thicknesses, but within the scope of the present embodiments, the resulting surface may be considered substantially flat or planar. 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 support, and particularly preferably less than 20% or 10% of the maximum thickness of the coated support.

[0018] 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 may provide an additional physical barrier to the cells if the cells tend to move away from the sample surface. This physical barrier may enhance the effectiveness of the coating that reduces sample adhesion 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 adhered to the sample surface. The carrier may include a wall located between the sample surface and the edge of the carrier for promoting adhesion of adherent samples to the sample surface and hindering movement of adherent samples away from the sample surface. To achieve this purpose, the geometry of the wall may be sufficient to hinder movement or adhesion of the sample on surfaces other than the sample surface, but it may also be advantageous if the adherent sample has weaker adhesion to the wall than to the sample surface during use.

[0019] The carrier can advantageously be positioned or positionable on a substrate for receiving a sample adhered to the substrate. In this case, when the carrier is positioned on the substrate, the peripheral region (eg, wall) is preferably located between the sample surface and the substrate.

[0020] The peripheral region preferably completely surrounds the sample surface, surrounds the carrier boundary in a continuous ring or annular shape, and preferably extends from the carrier edge to the outer boundary of the sample surface. Particularly preferably, in embodiments comprising a wall, the wall surrounds or encloses the sample surface.

[0021] Advantageously, the coating is applied to the peripheral region of the carrier and is configured to reduce or prevent sample adhesion to the peripheral region, such that adherent samples adhere less strongly to the peripheral region than to the sample surface. Thus, the coating applied to the peripheral region may be referred to as a sample-repelling coating, a non-adhesive 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.

[0022] The coating applied to the peripheral area may be a non-ionic surfactant. In a preferred embodiment, the peripheral area may be coated with one or more of Pluronic F-127, Poly-HEMA, or other Poloxamer.

[0023] The substrate or body of the carrier (to which the coating is applied) can be manufactured in any convenient manner. In a preferred embodiment, the substrate is manufactured using a photolithographic process. In such a case, the substrate, including the peripheral border below its peripheral region, can comprise a photoresist (such as SU-8). A second photolithographic step can then be used to selectively treat or coat the central portion of the carrier substrate, for example with a metal layer (such as a magnetic layer or a gold layer).

[0024] Alternatively, the base or body of the carrier may be formed using different materials for the central portion below the sample surface and for the peripheral portion below the peripheral region.

[0025] When the peripheral region of the carrier comprises a photoresist material such as SU-8, it can be first treated with an oxygen plasma and then coated with silane-terminated PEG molecules, such that the silane is covalently bound to the oxygen plasma-treated surface and the PEG molecules form a release coating.

[0026] Providing a non-sample-adherent peripheral region advantageously reduces the likelihood of sample cells migrating between adjacent carriers during use because the peripheral region creates a spatial separation between the sample surfaces of adjacent carriers when the carriers are positioned on the substrate. If the carriers are positioned on the substrate consistently in an overlapping manner, with the edge of one carrier overlapping the edge of another (e.g., as might occur if multiple carriers are deposited or magnetically manipulated onto the substrate during an assay), the peripheral region also increases the chance that the sample surface of each carrier will remain visible from above during analysis.

[0027] In embodiments of the present invention that include a wall, the geometry of the wall can help confine adherent samples to the sample surface, but geometric effects alone may not be sufficient. Therefore, at least a portion of the surface of the wall can advantageously include a surface treatment (such as a coating), for example, a coating that reduces sample adhesion, such that adherent samples adhere less strongly to the wall than to the sample surface. For example, the wall or a portion of the wall can be coated with a nonionic surfactant. The wall or a portion of the wall can be coated with Pluronic F-127, poly-HEMA, or other poloxamers. When the wall comprises a photoresist material (such as SU-8), the wall can be treated with oxygen plasma and then coated with silane-terminated PEG molecules, such that the silane covalently binds to the oxygen plasma-treated surface and the PEG molecules form a release coating.

[0028] Alternatively or in addition to providing a coating that reduces sample adhesion on the peripheral area, the sample surface may include a coating configured so that the adherent sample has a stronger adhesion to the sample surface than to the peripheral area. This helps to confine the adherent 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 to or adhere to the test sample. The sample surface may include a charged coating. This may be particularly preferred when the test sample itself is charged. For example, cells typically have a membrane potential between negative 40 and negative 80 millivolts. Providing a coating that defines a positively charged surface may promote the attachment of the test sample to the charged surface of the carrier. It has been found that providing a charged surface, and preferably a positively charged surface, can advantageously achieve more effective cell adhesion. The coating may include a charged polymer. 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.

[0029] The gold cap can be formed only in the region of the carrier forming the sample surface. If an adhesion-promoting coating is then applied to the gold layer, only the sample surface of the carrier is biofunctionalized and, therefore, only the sample surface of the carrier tends to receive the test sample, while the surrounding area (which does not include the functionalized gold cap) does not receive the test sample.

[0030] The polymer (charged polymer) can be covalently bound to a gold cap forming a sample surface. The sample surface suitable for receiving the test sample can include a gold cap layer to which the polymer is covalently bound via thiol groups. This advantageously ensures that each polymer adsorbed to the surface has the same orientation. Alternatively, the polymer can be adsorbed to the gold cap of the support via van der Waals forces.

[0031] The charged polymer may be a polymer comprising positively charged carrying groups. The polymer may be polyornithine or poly-d-lysine. The polymer may be a polyelectrolyte.

[0032] Alternatively or additionally, the coating on the sample surface may include multiple ligands. The multiple ligands may include antibodies. When the test sample to be detected is a cell, the multiple ligands may include antibodies that specifically bind to cell receptors (such as integrins).

[0033] When the test sample is a cell, the sample surface coating may alternatively or additionally include an extracellular matrix protein. The extracellular matrix protein can be a protein selected to increase or enhance cell adhesion. The protein can be collagen, laminin, or Matrigel, a mixture of proteins secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.

[0034] In a preferred embodiment, the sample surface may be substantially flat and preferably parallel to the substrate when the carrier is positioned on the substrate. The carrier or each carrier may advantageously include a body or base portion, which is preferably layered and may be a high aspect ratio shape, such that its lateral dimensions (or 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 portion (e.g., one of the larger surfaces of the high aspect ratio shape) may then form or carry the sample surface. A peripheral region is preferably also provided on the upper surface of the base portion, substantially in the same plane (or coplanar) as the sample surface. Alternatively, a wall may extend upwardly from the upper surface of the base portion, adjacent to or spaced apart from the sample surface. As described above, the sample surface and the peripheral region may carry surface treatments (such as coatings) of different thicknesses, so that they may not be completely in the same plane.

[0035] The carrier is preferably a planar particle. The carrier particle preferably includes a planar first side and a planar second side, the second side being arranged parallel to the first side on the opposite side of the carrier particle. The sample surface and the peripheral region are preferably provided on the same side of the carrier (e.g., both on the first side), while the other side may be attached to a release layer, for example. Due to the presence of the peripheral region, the sample surface may not occupy the entire surface of the carrier and may not extend to the edge of the carrier.

[0036] To achieve the desired effect of limiting sample movement from the edge of the carrier and inhibiting sample migration to other carriers, at least a portion of the peripheral region preferably has a width greater than 1 micron and less than 50 microns, or greater than 5 microns and less than 30 microns, preferably greater than 8 microns and less than 25 microns. For example, the peripheral region may have a width of 10 microns. Preferably, the peripheral region has a uniform width around the sample surface.

[0037] To achieve the desired geometric effect of the wall, at least part of the wall may have a height above the sample surface greater than 1, 2, or 3 microns, and less than 50, 20, or 15 microns. Furthermore, at least part of the wall may have a lateral thickness or maximum lateral thickness greater than 1 micron and less than 50 microns, preferably greater than 5 microns and less than 40 microns.

[0038] To adjust the effect of the wall on limiting or constraining adherent samples, at least a portion of the wall may have a rectangular cross-section, or a flared or circular 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, such that the cross-section of the wall further 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 may improve the constraint on adherent samples. Alternatively, the cross-section of the wall may be flared, such that the cross-section of the wall further away from the sample surface is smaller than the cross-section of the wall closest to the sample surface. This may allow adherent cells to more easily reach the sample surface of the matrix portion. Preferably, the wall has the same cross-section over its entire length.

[0039] A flared wall, where the top of the wall is thicker than the bottom, or a wall that overhangs the sample surface, can advantageously provide a mechanical barrier that prevents the migration of cells that have left the sample surface. Alternatively, a flared wall, where the top of the wall is thinner than the bottom of the wall, can advantageously allow any cells that may have adhered to the wall to migrate downward to the sample surface. Thus, the shape of the wall can be optimized for different cell or sample types, as well as for different wall and sample surface coatings.

[0040] Advantageously, the support may have a lateral dimension (preferably the smallest lateral dimension) greater than 5 microns and less than 300 microns, preferably greater than 10 microns and less than 200 microns.

[0041] The portion of the carrier below the peripheral region can be manufactured by any suitable method, but advantageously, for convenience and efficiency, it can include material(s) 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 or in the same process as the portion of the substrate below the sample surface. These portions of the substrate of the carrier can be made of the same material(s), extending continuously between the peripheral region and below the sample surface.

[0042] For example, the base portion may conveniently be manufactured using photolithographic processes, in which case the magnetic material may also be incorporated into the carrier.

[0043] In a preferred embodiment, the peripheral region is only the region of the coating of the base part of the carrier.

[0044] The peripheral region of the carrier can be manufactured using nanoimprint or microimprint lithography.

[0045] In a preferred embodiment, the sample surface is only the area of ​​the coating of the base portion of the carrier.

[0046] In one embodiment, in particular if the carrier is manufactured using photolithography, at least the base portion of the carrier below the peripheral area may comprise a structural photopolymer, preferably a photoresist, SU-8 or AZ-10nXT. Alternatively or additionally, at least the base portion below the peripheral area may comprise a polymer, such as a thermoplastic polymer, preferably PDMS.

[0047] The walls of the carrier may be manufactured by any suitable method, but advantageously, for convenience and efficiency, the walls may comprise a material also present in the base portion, and the walls are preferably manufactured using the same process used to manufacture the base portion. For example, the walls and base portion may conveniently be manufactured using a photolithographic process, in which case the magnetic material may also be incorporated into the carrier.

[0048] The walls can be fabricated using nanoimprint lithography or microimprint lithography.

[0049] In one embodiment, in particular if the carrier is manufactured using exposure lithography, the wall may comprise a structural photopolymer, preferably photoresist, SU-8 or AZ-10nXT. Alternatively or additionally, the wall may comprise a polymer, such as a thermoplastic polymer, preferably PDMS.

[0050] In a further aspect, the present invention can advantageously provide a carrier system for detection, comprising a plurality of carriers. In the carrier system, the carriers are preferably initially locked to a substrate, for example, by a release layer. The carriers on the substrate can then be exposed to a biocompatible solution in which an adherent detection sample (such as adherent cells) is suspended, allowing the adherent sample to adhere to the sample surface of the carrier. The release layer can be activated by the biocompatible solution or dissolved in the biocompatible solution, allowing the carriers carrying the adherent sample to be released from the substrate into the solution. Advantageously, during detection, the adherent sample can be carried on the sample surface of each carrier and is limited from bridging or crossing from the sample surface to the substrate by a peripheral area (e.g., a wall) separating the sample surface from the edge of the carrier.

[0051] In a further preferred embodiment, the substrate between the carriers may additionally receive a surface treatment or be coated in order to reduce adhesion to adherent samples and thereby further reduce any risk that the sample may bridge or cross from the carrier to the substrate.

[0052] For example, during manufacture of the carrier system, a release layer is preferably applied to the entire substrate, including the (subsequently formed) regions between carriers. In one embodiment, the release layer between carriers can be removed after at least the carrier matrix has been formed, and the underlying substrate is treated or coated to reduce cell adhesion.

[0053] Applying such a surface treatment or coating to the release layer itself may be unsuitable because the release layer typically dissolves in a biocompatible solution when the adherent sample is loaded onto the sample surface of the carrier.

[0054] The carrier can advantageously be magnetic or comprise a magnetic material. In such a case, the carrier can still be fixed to the substrate by an applied magnetic field even after the release layer has dissolved, allowing more time for adherent samples to adhere to the sample surface.

[0055] In yet a further aspect, the present invention may advantageously provide a method for performing a test using the above-described carrier or carrier system. The method may advantageously include the following steps: providing a plurality of carriers positioned on a substrate, introducing a biocompatible aqueous solution carrying adherent samples to the carriers and the substrate such that the adherent samples adhere to the sample surface of the carriers rather than to the peripheral area of ​​the carriers, and releasing the carriers from the substrate. The carriers may be fixedly positioned on the substrate by a release layer between the carriers and the substrate, the release layer being activated by the biocompatible aqueous solution to release the carriers. Additionally or alternatively, the carriers may be magnetic or include a magnetic material and may then be fixedly positioned on the substrate by an applied magnetic field and released from the substrate by changing or removing the applied magnetic field.

[0056] In yet a further aspect of the present invention, it should be noted that the carriers of the present invention are preferably magnetically manipulable (e.g., throughout the various stages of the assay). Thus, after release from the substrate, the solution being assayed can include a plurality of carriers carrying adherent samples. In solution, the carriers can move closer to one another, and it is important to prevent adherent samples from migrating from one carrier to another. While the carriers are in solution, the sample repulsion effect of the peripheral region surrounding the adherent samples on each carrier can advantageously help keep the sample surfaces of different carriers spaced apart from one another, thereby reducing the risk of adherent samples migrating from one carrier to another.

[0057] In some cases, individual supports may include a readable code or be otherwise labeled so that individual supports or groups of supports can be identified during testing. If adherent samples are placed on individual supports at the start of the test, it is essential to ensure that these samples do not migrate from one support to another. A coated peripheral area in solution surrounding samples on supports that may migrate adjacent to or past other supports can advantageously prevent sample migration between supports.

[0058] For example, a readable code (such as a barcode or a QR code) can be applied to the surface of the carrier, or it can include a hole or several holes that are defined to pass through the matrix portion of the carrier or in the matrix portion of the carrier. Preferably, the code can be located in the peripheral area. This advantageously prevents the code from reducing the area of ​​the sample surface available for cell attachment. Preferably, the wall includes a readable code. Advantageously, the readable code included in the wall prevents the code from reducing the area of ​​the sample surface available for cell attachment. The code can partially penetrate into the wall. The code can penetrate through the wall to the carrier matrix. The readable code can include several dots, short lines, or dots and short lines. The code can include several solid lines. The code can be formed by photolithography.

[0059] The readable code can take a variety of forms as a marking on a carrier that encodes information identifying the carrier on which the readable code is applied. When imaged, the pattern in a given carrier can be analyzed for its encoded information and, therefore, used to identify that particular carrier. The code is preferably readable by image analysis of a microscope image.

[0060] Furthermore, magnetically manipulable supports can often be manipulated onto a substrate during detection (e.g., for imaging or analysis). When the support is placed on a substrate in this manner, the coated peripheral region on the support can advantageously prevent adherent samples from migrating from the support to the substrate, or from one support to another.

[0061] As mentioned above, the sample surface and the peripheral region of the carrier implementing the present invention can be coplanar, or it can alternatively not be coplanar. One option is to make the peripheral region raised relative to the sample surface. This aspect of the present invention can be advantageously related to further aspects of the present invention discussed below.

[0062] In this further aspect, the present invention can therefore provide a carrier for adhering to a sample for detection. The carrier can include a sample surface for receiving the adherent sample, and a wall located between the sample surface and an edge of the carrier, configured to promote adhesion of the adherent sample to the sample surface and hinder movement of the adherent sample away from the sample surface. To achieve this, the geometry of the wall can be sufficient to hinder movement or adhesion of the sample to surfaces other than the sample surface, but it may also be advantageous if the adherent sample has stronger adhesion to the sample surface than to the wall during use.

[0063] The carrier can advantageously be positioned on a substrate for receiving a test sample adhered to the wall. In such a case, when the carrier is positioned on the substrate, the wall is preferably located between the sample surface and the substrate.

[0064] Particularly preferably, the wall surrounds or encloses the sample surface.

[0065] In a preferred embodiment, the carrier can form part of a carrier system, wherein multiple carriers are provided on a substrate and are preferably locked to the substrate by, for example, a release layer. The carriers on the substrate can then be exposed to a biocompatible solution, wherein an adherent test sample (such as adherent cells) is suspended in the biocompatible solution, allowing the adherent sample to adhere to the sample surface of the carrier. The release layer can be activated by or dissolved in the biocompatible solution, allowing the carrier carrying the adherent sample to be released from the substrate into the solution. Advantageously, the adherent sample can be carried on the sample surface of each carrier and is restricted from bridging or crossing from the sample surface to the substrate by the wall.

[0066] The carrier can advantageously be magnetic or comprise a magnetic material. In this case, even if the release layer has dissolved, the carrier can still be fixed to the substrate by an applied magnetic field, allowing more time for adherent samples to adhere to the sample surface.

[0067] In a preferred embodiment, the sample surface is substantially planar and preferably parallel to the substrate when the carrier is positioned on the substrate. The or each carrier may advantageously comprise a base portion, which is preferably laminar and may be a high-aspect-ratio shape, such 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. The 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 the wall may extend upwardly from the upper surface of the base portion, adjacent to or spaced from the sample surface.

[0068] While the geometry of the wall may be sufficient to confine adherent samples to the sample surface, for some types of adherent 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) that allows adherent samples to adhere more strongly to the sample surface than to the wall. For example, the sample surface may include a gold capping layer to which a polymer is covalently bound via thiol groups. The polymer may include positively charged groups. The polymer may be polyornithine or poly-d-lysine. The polymer may be a polyelectrolyte.

[0069] Alternatively or additionally, at least a portion of the surface of the wall may include a surface treatment (such as a coating) such that adherent samples 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 poloxamer. When the wall comprises a photoresist material (such as SU-8), the wall may be treated with oxygen plasma and then coated with silane-terminated PEG molecules such that the silane covalently binds to the oxygen plasma-treated surface and the PEG molecules form a release coating.

[0070] To achieve the desired geometric effect of the wall, at least part of the wall may have a height above the sample surface greater than 1 micron and less than 50 microns, preferably greater than 2 microns and less than 20 microns, or greater than 3 microns and less than 15 microns. In addition, at least part of the wall may have a lateral thickness or maximum lateral thickness greater than 1 micron and less than 50 microns, preferably greater than 5 microns and less than 40 microns.

[0071] To adjust the effect of the wall on confining or restraining the adherent sample, at least a portion of the wall may have a rectangular cross-section, or a flared or circular 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, such that the cross-section of the wall further 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 may improve the restraint of the adherent sample. Alternatively, the cross-section of the wall may be flared, such that the cross-section of the wall further away from the sample surface is smaller than the cross-section of the wall closest to the sample surface. This may allow adherent cells to more easily reach the sample surface of the matrix portion. Preferably, the wall has the same cross-section over its entire length.

[0072] A flared wall, where the top of the wall is thicker than the bottom, or a wall that overhangs the sample surface, can advantageously provide a mechanical barrier that prevents the migration of cells that have left the sample surface. Alternatively, a flared wall, where the top of the wall is thinner than the bottom of the wall, can advantageously allow any cells that may have adhered to the wall to migrate downward to the sample surface. Thus, the shape of the wall can be optimized for different cell or sample types, as well as for different wall and sample surface coatings.

[0073] Advantageously, the support may have a lateral dimension (preferably the smallest lateral dimension) greater than 5 microns and less than 300 microns, preferably greater than 10 microns and less than 200 microns.

[0074] The walls of the carrier may be manufactured by any suitable method, but advantageously for convenience and efficiency the walls may comprise a material also present in the base portion, and the walls are preferably manufactured using the same process used to manufacture the base portion. For example, the walls and base portion may conveniently be manufactured using a photolithographic process, in which case the magnetic material may also be incorporated into the carrier.

[0075] The walls can be fabricated using nanoimprint lithography or microimprint lithography.

[0076] In one embodiment, in particular if the carrier is manufactured using exposure lithography, the wall may comprise a structural photopolymer, preferably a photoresist, SU-8 or AZ-10nXT. Alternatively or additionally, the wall may comprise a polymer, such as a thermoplastic polymer, preferably PDMS.

[0077] In a further aspect, the present invention can advantageously provide a carrier system for detection, the carrier system comprising a plurality of carriers. In the carrier system, the carriers are preferably provided to be locked to a substrate via a release layer as described above. The substrate surface to which the carriers are locked is preferably flat.

[0078] In yet a further aspect, the present invention may advantageously provide a method for performing a test using a carrier or carrier system as described above. The method may advantageously include the following steps: providing a plurality of carriers positioned on a substrate, introducing a biocompatible aqueous solution carrying adherent samples to the carriers and the substrate, causing the adherent samples to adhere to the sample surface of the carriers, and releasing the carriers from the substrate. The carriers may be fixedly positioned on the substrate by a release layer between the carriers and the substrate, the release layer being activated by the biocompatible aqueous solution to release the carriers. Additionally or alternatively, the carriers may be magnetic or include a magnetic material and may then be fixedly positioned on the substrate by an applied magnetic field and released from the substrate by changing or removing the applied magnetic field.

[0079] In yet a further aspect of the invention, the carriers of the invention are preferably magnetically manipulable (e.g., throughout the various stages of the assay). Thus, after release from the substrate, the solution being assayed can include a plurality of carriers carrying adherent samples. In solution, the carriers can move closer to one another, and it is important to prevent adherent samples from migrating from one carrier to another. The geometric effect of the walls on each carrier, which preferably surround the adherent samples, can advantageously help keep sample surfaces on different carriers spaced apart from one another while the carriers are in solution, and reduce the risk of adherent samples migrating from one carrier to another.

[0080] In some cases, individual carriers can be labeled with a readable code (such as a barcode) or other marking method so that individual carriers or groups of carriers can be identified during the assay. If adherent samples are placed on individual carriers at the beginning of the assay, it is essential to ensure that these samples do not migrate from one carrier to another. A wall surrounding the samples on the carriers (which may move close to or past other carriers) in solution can advantageously prevent sample migration between carriers.

[0081] The carrier may include a readable code applied to the sample surface of the carrier. Alternatively, the readable code may include a hole, several holes, an area or an area of ​​different materials that is defined to penetrate the matrix portion of the carrier or within the matrix portion of the carrier. Preferably, the wall includes the readable code. Advantageously, the readable code included in the wall prevents the code from reducing the area of ​​the sample surface available for cell attachment. The readable code may include several dots, dashes or dots and dashes. The code may include several solid lines. The code may partially penetrate into the wall. The code may penetrate through the wall to the carrier matrix. The code may be formed by photolithography.

[0082] The readable code can take a variety of forms as a marking on a carrier, encoding information that identifies the carrier to which the code is applied. When imaged, the pattern in the wall or matrix of a given carrier can be analyzed for its encoded information and, therefore, used to identify that particular carrier. The code is preferably readable by image analysis of a microscope image.

[0083] Furthermore, during detection, magnetically manipulable carriers can often be manipulated onto a substrate (e.g., for imaging or analysis). When the carrier is placed on a substrate in this manner, the wall can advantageously prevent the adhered sample from moving from the carrier to the substrate, or from one carrier to another. Furthermore, as described above, carriers moving within a solution during detection may move close to or even come into contact with other carriers, and the wall can advantageously prevent the movement of samples between such carriers. These effects can only be achieved by placing the wall on the carrier itself. In the prior art, walls have been used to position or confine adhered samples on a substrate, but because the walls of the prior art are locked to the substrate, and therefore, after the carrier (such as a magnetically manipulable carrier) is moved or manipulated to a new substrate during detection, the wall has no effect.

[0084] Furthermore, when the carrier is driven through the test medium by an external magnetic field, the test sample carried by the carrier is subjected to shear forces. These forces may be detrimental to the test sample, and the presence of a wall adjacent to the test sample on the sample surface of the carrier can advantageously reduce the shear forces experienced by the test sample.

[0085] The present invention is defined by the claims. However, a non-exhaustive, non-limiting list of examples is provided below. Any one or more features of these examples may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0086] Example (EX): EX1. A carrier for adhering to a wall to detect samples, comprising a sample surface for receiving the adhering sample and a peripheral area surrounding the sample surface and located between the sample surface and the edge of the carrier, wherein the carrier includes a surface treatment so that the adhesion of the adhering sample to the peripheral area is weaker than the adhesion to the sample surface.

[0087] EX2. A carrier according to example EX1, wherein the carrier is removably positionable on a substrate for receiving an adherent detection sample during use, wherein when the carrier is positioned on the substrate, the peripheral area is between the sample surface and the substrate, preferably surrounding the sample surface.

[0088] EX3. A carrier according to any of the preceding items, wherein when the carrier is positioned on a substrate, the height difference between the sample surface and the peripheral area above the substrate is less than 10% of the maximum thickness of the carrier, and preferably less than 1% or 0.1% of the maximum thickness of the carrier.

[0089] EX4. A carrier according to any of the preceding items, wherein the thickness of the carrier in the region of the sample surface differs from the thickness of the carrier in the surrounding region by less than 10%, and preferably by less than 1% or 0.1%.

[0090] EX5. A carrier according to any of the preceding items, wherein the peripheral region is substantially flat and preferably parallel to the substrate when the carrier is positioned on the substrate.

[0091] EX6. A carrier according to any of the preceding items, wherein the sample surface is substantially flat and preferably parallel to the substrate when the carrier is positioned on the substrate.

[0092] EX7. A carrier according to any of the preceding items, wherein the carrier comprises a base portion, and an upper surface of the base portion forms the sample surface and the peripheral area.

[0093] EX8. A carrier according to any of the preceding items, wherein the peripheral area comprises a coating that reduces sample adhesion.

[0094] EX9. The carrier of Example EX8, wherein the coating on the peripheral region comprises a nonionic surfactant.

[0095] EX10. The carrier of example EX8 or EX9, wherein the coating on the peripheral region comprises one or more selected from Pluronic F-127, poly-HEMA, PEG polymers, and poloxamers.

[0096] EX11. A carrier according to any of the preceding items, wherein the sample surface comprises a sample adhesion promoting coating.

[0097] EX12. The carrier of example EX11, wherein the sample surface comprises a biofunctionalized coating suitable for receiving adherent samples.

[0098] EX13. The carrier of example EX11 or EX12, wherein the sample surface comprises a charged polymer.

[0099] EX14. The carrier according to any of the preceding items, 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.

[0100] EX15. The carrier according to any of the preceding items, wherein the carrier has a lateral dimension, preferably a smallest lateral dimension, greater than 5 micrometers and less than 300 micrometers, or preferably greater than 10 micrometers and less than 200 micrometers.

[0101] EX16. A carrier according to any of the preceding items, wherein the carrier comprises a magnetic material.

[0102] EX17. A vector system for detection, comprising a plurality of vectors as defined in any one of Examples EX1 to EX16.

[0103] EX18. A method for performing detection using the carrier defined in any one of Examples EX1 to EX16 or the carrier system defined in Example EX17, the method comprising the following steps: providing a plurality of carriers positioned on a substrate, introducing a biocompatible aqueous solution carrying adherent samples into the carriers and the substrate such that the adherent samples adhere to the sample surface of the carriers rather than the surrounding area, and releasing the carriers from the substrate.

[0104] EX19. The method of performing a detection according to example EX18, wherein the carrier is fixed 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.

[0105] EX20. A method of performing detection according to example EX18 or EX19, wherein the carrier is magnetic or includes a magnetic material and is fixedly positioned on the substrate by an applied magnetic field and is released from the substrate by changing or removing the applied magnetic field.

[0106] EX21. A carrier for detecting adhered samples, comprising a sample surface for receiving adhered samples when in use and a wall located between the sample surface and an edge of the carrier.

[0107] EX22. A carrier according to example EX21, wherein the adherent sample has stronger adhesion to the sample surface than to the wall during use.

[0108] EX23. A carrier according to example EX21 or EX22, wherein during use the carrier is removably positionable on a substrate for receiving a wall-adherent detection sample, wherein when the carrier is positioned on the substrate, the wall is located between the sample surface and the substrate.

[0109] EX24. A carrier according to any one of examples EX21 to EX23, wherein the wall surrounds the sample surface.

[0110] EX25. The carrier of any of examples EX21 to EX24, wherein the sample surface is substantially flat and preferably parallel to the substrate when the carrier is positioned on the substrate.

[0111] EX26. A carrier according to any one of examples EX21 to EX25, wherein the carrier includes 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, adjacent to or spaced from the sample surface.

[0112] EX27. The carrier of any one of examples EX21 to EX26, wherein at least a portion of the sample surface comprises a coating such that the adherent sample adheres more strongly to the sample surface than to the wall.

[0113] EX28. The carrier of any one of examples EX21 to EX27, wherein a surface of the wall at least partially comprises a coating such that adhesion of the adherent sample to the wall is weaker than adhesion to the sample surface.

[0114] EX29. The carrier of any of examples EX21 to EX28, wherein at least a portion of the wall has a height above the sample surface that is greater than 1 micrometer and less than 25 micrometers.

[0115] EX30. The carrier of any of examples EX21 to EX29, 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.

[0116] EX31. A carrier according to any one of examples EX21 to EX30, wherein at least a portion of the wall has a rectangular cross-section, or has a flared or circular cross-section.

[0117] EX32. The carrier according to any one of examples EX21 to EX31, wherein the carrier has a lateral dimension, preferably a smallest lateral dimension, greater than 5 micrometers and less than 300 micrometers, or preferably greater than 10 micrometers and less than 200 micrometers.

[0118] EX33. A carrier according to any one of examples EX21 to EX32, wherein the carrier comprises a magnetic material.

[0119] EX34. A carrier according to any one of examples EX21 to EX33, wherein the carrier comprises a base portion, an upper surface of which forms the sample surface, and wherein the wall comprises a material also present in the base portion and is preferably manufactured using a process also used to manufacture the base portion.

[0120] EX35. The carrier of any one of examples EX21 to EX34, wherein the carrier comprises a base portion, an upper surface of which forms the sample surface, and wherein the wall and the base portion are manufactured using a photolithographic process.

[0121] EX36. The carrier of any one of examples EX21 to EX35, wherein the wall comprises a structured photopolymer, preferably a photoresist, SU-8, or AZ-10nXT.

[0122] EX37. A carrier according to any one of examples EX21 to EX36, wherein the wall comprises a polymer, such as a thermoplastic polymer, preferably PDMS.

[0123] EX38. The carrier according to any one of examples EX21 to EX37, wherein the sample surface comprises a gold capping layer to which the polymer is covalently bound via thiol groups.

[0124] EX39. A vector system for detection, comprising a plurality of vectors as defined in any one of Examples EX21 to EX38.

[0125] EX40. A method for performing a test using the carrier defined in any one of Examples EX21 to EX38 or the carrier system defined in Example EX39, the method comprising the steps of providing a plurality of carriers positioned on a substrate, introducing a biocompatible aqueous solution carrying adherent samples into the carriers and the substrate such that the adherent samples adhere to the sample surface of the carriers, and releasing the carriers from the substrate.

[0126] EX41. A method for performing detection according to example EX40, wherein the carrier is fixedly positioned 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.

[0127] EX42. The detection method of example EX40 or EX41, wherein the carrier is magnetic or includes a magnetic material and is fixedly positioned on the substrate by an applied magnetic field and is released from the substrate by changing or removing the applied magnetic field. DETAILED DESCRIPTION

[0128] Specific embodiments of the present invention will now be described by way of example with reference to the accompanying drawings, in which Figure 1 is a schematic cross-sectional view of three carriers locked on a substrate according to a first embodiment of the present invention; Figure 2 For example Figure 1 A schematic cross-sectional view of the carrier shown after release from the substrate; Figure 3 For example Figure 1 and Figure 2 a schematic three-quarter view of the carrier shown; Figure 4 A micrograph of a carrier array locked to a substrate for practicing the present invention; Figure 5Photomicrographs of multiple carriers carrying adherent cells and suspended in a test solution for the implementation of the present invention; Figure 6 is a schematic cross-sectional view of three carriers attached to a substrate via a release layer according to a second embodiment of the present invention (the vertical dimensions are exaggerated for clarity); Figure 7 for Figure 6 A schematic cross-sectional view of the carrier shown after release from the substrate; Figure 8 The present invention is similar to Figure 6 Schematic cross-sectional views of five alternative embodiments of the carrier wall having different cross-sectional shapes after release from the substrate; Figure 9 To support Figure 6 and Figure 7 a photomicrograph plan view of the substrate of the carrier array shown; and Figure 10 This is a micrograph of a carrier carrying adherent cells and suspended in a detection solution as in the second embodiment.

[0129] The carrier according to the first embodiment of the present invention is shown in Figure 1 、 Figure 2 and Figure 3 In. Figure 1 As shown, carrier 100 is initially provided for detection and is locked onto substrate 12 in the form of a silicon chip via a release layer 14, which may include dextran. Depending on the detection application, the substrate is sized to support a plurality of carriers, ranging from approximately 1,000 to 500,000. Each carrier is suitable for receiving one or more adherent cells.

[0130] The fabrication process for each carrier 100 is as follows: a release layer 14 is spin-coated onto a silicon substrate 12. A square layer 16 of structured photopolymer is then deposited onto the release layer. The shape of the photopolymer is defined by photolithography and is approximately 1.5 microns thick, with each side of the square measuring 120 microns. Thus, the shape of the photopolymer (which can be SU-8) is a flat or high-aspect-ratio rectangular parallelepiped with relatively large, square upper and lower surfaces. The photopolymer square forms the base or body of the carrier.

[0131] Using photolithographic techniques as described in WO 2020 / 099846, a multilayer magnetic heterostructure 20 is deposited on a square central portion of the upper surface of the photopolymer layer 16. The magnetic heterostructure is deposited only on the central portion of the photopolymer layer to define the shape of the sample surface.

[0132] Next, a functionalized gold cap 22 is deposited on the multilayer magnetic heterostructure 20, followed by a polymer layer 24 to which the target adherent sample can easily adhere. This polymer layer 24 defines the sample surface 25 of the carrier that receives the test sample during the test. The sample surface is located in the center of the carrier substrate and has a square shape with a side length of approximately 100 microns.

[0133] In this embodiment, the peripheral region 28 surrounding the sample surface is 10 microns wide.

[0134] The polymer of polymer layer 24 includes thiol groups and is applied by immersing the carrier and substrate in a solution containing the polymer. The polymer covalently bonds to the gold cap 22 of the carrier via the thiol groups. The solution includes a non-aqueous solvent (such as ethanol). Since the release layer is insoluble in such non-aqueous solvents, the carrier remains locked to the substrate when immersed in the solution containing the polymer.

[0135] The cumulative thickness of the magnetic heterostructure, gold cap and polymer layer is less than 1 micron and typically several hundred nanometers, depending on the thickness of the attached polymer layer. Thus, the upper surface of the completed carrier is substantially flat or planar.

[0136] Each carrier can include a readable code (such as a barcode). A predefined code is assigned to each carrier to identify the carrier. For example, the predefined code can point to a specific adherent cell received on the carrier, or a specific adherent cell and a specific reagent to which the adherent cell has 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 can be defined in the peripheral region of the carrier. In an example, the code comprises a number of dots or short lines defined in the peripheral region by photolithography (e.g., by a single exposure of a SU-8 substrate to create holes or indentations).

[0137] The carrier of the present invention can also be implemented by forming a magnetic heterostructure that spans the entire carrier and then defining the area and shape of the sample surface 25 as a portion of the upper surface of the magnetic heterostructure. This can be achieved by depositing a gold cap or polymer coating on a selected area of ​​the carrier surface, where the selected area is surrounded by a peripheral area 28 that extends to the edge of the carrier surface. Thus, the sample surface occupies a relatively central position on the carrier surface, while the peripheral area 28 extends completely around the sample surface, spanning the area between the sample surface and the edge of the carrier.

[0138] In a further preferred embodiment, the sample surface 25 is approximately 100 microns square, and the width of the peripheral region between the edge of the carrier and the edge of the sample surface is approximately 20 microns.

[0139] The sample surface 25 and the peripheral area 28 may be coated differently, or may be different materials or treated with different materials, such that the test sample adheres more strongly to the sample surface 25 than to the peripheral area 28 .

[0140] Thus, for example, after forming the sample surface 25 as described above, the peripheral region 28 of the upper surface of the photopolymer layer 16 is coated with an adhesion-reducing coating material to reduce or prevent adhesion of the test sample (e.g., sample cells) to the peripheral region 28 of the carrier 10. The coating of the peripheral region can be formed by depositing a nonionic surfactant (such as Pluronic F-127) in a band surrounding the central sample surface. For example, the sample surface can be coated with a gold cap, on which the low-adhesion surface treatment will not be formed.

[0141] In various embodiments, rather than depositing the gold cap 22 by photolithography only on the central sample surface 25, the gold cap 22 can be formed to cover the entire upper surface of the carrier 100, and the corresponding coating or coatings defining the sample surface can be applied only to defined areas on the gold cap. For example, the gold cap can be functionalized only on the sample surface area in the center of the carrier, while the adhesion-reducing coating can be applied to the exposed portion of the gold cap around the perimeter of the carrier to surround the sample surface.

[0142] In other embodiments, a coating other than the polymer coating 24 described above can be used to coat the sample surface. For example, an adhesion-promoting sample surface coating can include a plurality of 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. In addition to applying a coating to the sample surface, the method can include modifying the physical surface of the sample surface to be particularly suitable for receiving adherent cells, such as by modifying a gold cap.

[0143] In other embodiments, various adhesion reducing coatings can be used to reduce the adhesion of the sample to the peripheral region 18, 28. For example, the peripheral region can be coated with poly-HEMA or other poloxamers.

[0144] Figure 4 A micrograph of a carrier array locked on a substrate is shown. Any number of carriers can be fixed to the substrate as required for any detection application.

[0145] The final step in the carrier system manufacturing process is sterilization (not shown). To sterilize the carrier system, the carrier array locked to the substrate is placed in a sterilization package, sealed, and then placed in an oven at 120 degrees Celsius for 4 hours. This sterilizes it and produces a ready-to-use sample that can be opened in a sterile cell culture environment.

[0146] Figure 5 To implement the present invention, several carriers were used in adherent cell assays, with the carriers suspended in an assay solution. During imaging, the magnetic carriers were aligned to face the camera through appropriate adjustment of the external magnetic field, and the cells were stained to improve visibility. It can be seen that the cells adhered to the sample surface of each carrier, but not to the surrounding area surrounding the sample surface.

[0147] for Figure 5 To place cells on a sample surface on a carrier, the following steps are performed: a carrier system, such as a carrier array, on a silicon substrate is placed in a container and immersed in a biocompatible aqueous solution containing adherent cells. When the biocompatible aqueous solution contacts the release layer 14, the release layer dissolves and releases the carriers. If additional time is required for the cells to adhere to the carriers while on the substrate, an external magnetic field may be applied to maintain contact between the carriers and the substrate even after the release layer releases the carriers.

[0148] Once the cells adhere to the carrier, the carrier is released from the substrate and can move freely in the aqueous solution surrounding the carrier system. An external magnetic field can be used to apply a force to the carrier to manipulate and displace the carrier as desired to perform detection. Figure 5 As shown, a magnetic field can be used to align the carrier for photographic imaging.

[0149] The structure of the carrier 210 according to the second embodiment of the present invention is as follows Figure 6 and Figure 7 shown. Figure 6 Three carriers are shown locked onto a substrate 212 in the form of a silicon chip via a release layer 214. The substrate is sized to accommodate a plurality of carriers, ranging from approximately 1,000 to 500,000, depending on the assay application. Each carrier is adapted to receive one or more adherent cells.

[0150] The manufacturing process of the carrier(s) 210 is as follows: a release layer 214, which may include dextran, is spin-coated onto a silicon substrate 212. A square layer 216 of structured photopolymer is then deposited onto the release layer. The photopolymer shape is defined by photolithography and has a lateral dimension of approximately 1.5 microns thick along each side of the 160 micron square. Thus, the shape of the photopolymer (which may include SU-8) is a flat or high-aspect-ratio rectangular parallelepiped with relatively large square upper and lower surfaces. Upright walls 218 of photopolymer are then formed along the edges of the square layer 216, again by exposure photolithography. The walls are 7 microns high and 20 microns thick.

[0151] A surface treatment is then applied to at least a portion of the wall that reduces the adhesion of adherent samples to the wall. For example, coating can be accomplished by immersion in an ethanol solution containing Pluronic F127, which binds to the hydrophobic photopolymer comprising the wall, or by treating the wall surface with an oxygen plasma and immersing in an ethanol solution of silane-PEG ethanol, wherein the silane-PEG ethanol is covalently bound to the surface activated by the oxygen plasma. (In contrast, in preferred embodiments, the sample surface may include a gold layer, which may have a different hydrophobicity than the photopolymer of the wall and does not form surface -OH groups upon plasma treatment. Thus, a low-adhesion surface treatment may not be applied to the sample surface.) In some embodiments, it may be desirable to apply the low adhesion surface treatment to only specific portions of the wall. This can be achieved by masking. For example, a photoresist sacrificial mask can be patterned before applying the low adhesion surface treatment to block specific areas of the wall from being coated.

[0152] Alternatively, 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 specific uses of the carrier system where adhesion of the sample to the substrate may be an issue. When the carrier system is exposed to a biocompatible aqueous solution carrying adherent samples, the exposed release layer on the substrate will dissolve during use, and therefore, in order to apply a surface treatment between the carriers, the exposed release layer can be removed from the substrate and a surface treatment such as Pluronic 127 or silane-polyethylene glycol can be applied to the substrate between the carriers.

[0153] Then, on the upper surface of the photopolymer layer 216 within the walls 218 , a multilayer magnetic heterostructure 220 is deposited (as described in WO 2020 / 099846).

[0154] Next, a functionalized gold cap 222 is deposited on the multilayer magnetic heterostructure 220, followed by a polymer layer 224 to which the target adherent sample can easily adhere. The polymer layer 224 defines the sample surface of the carrier for receiving the test sample during the test, which is approximately 100 microns square.

[0155] The polymer of polymer layer 224 includes thiol groups and is applied by immersing the carrier and substrate in a solution containing the polymer. The polymer covalently bonds to the gold cap 222 of the carrier via the thiol groups. The solution includes a non-aqueous solvent (such as ethanol). Since the release layer is insoluble in such non-aqueous solvents, the carrier remains locked to the substrate when immersed in the solution containing the polymer.

[0156] The cumulative thickness of the magnetic heterostructure, gold cap, and polymer layer is less than 1 micrometer and typically several hundred nanometers, depending on the thickness of the attached polymer layer, so that the wall extends about 6 to 7 micrometers above the sample surface.

[0157] Each carrier can include a readable code (such as a barcode). A predefined code is assigned to each carrier to enable identification of the carrier. For example, a predefined code can identify a carrier carrying a specific adherent cell, as well as any specific reagents to which the adherent cell has been exposed as part of an assay. In other words, the code allows multiple carriers to perform multi-channel assays. The code can be defined in the wall of the carrier. In an embodiment, for example, a barcode consisting of dots and dashes, or solid lines can be included in a photolithographic pattern for the wall. This can be as simple as a number of dots, dashes, or solid lines, or more complex as an actual barcode. When imaging, the pattern in the wall of a given carrier can be used to analyze its barcode information.

[0158] As described above, in other embodiments, different shapes or cross-sections of the wall may be suitable for different cell or sample types, as well as different wall and sample surface coatings. Figure 8 Some examples are shown in .

[0159] Figure 8 The first two examples (from left to right) show flared walls that are thicker at the top than at the bottom, allowing the wall to overhang the sample surface. Meanwhile, the outer surface of the wall can be vertical, perpendicular to the carrier base, or flared outward. Such walls can advantageously provide a mechanical barrier to cell migration away from the sample surface and restrict cell migration toward the substrate between the carriers.

[0160] Alternatively, as Figure 8 As shown in the upper and lower right of Figure 1, a flared wall with a thinner top than the bottom of the wall can also be used. Such a wall shape can advantageously encourage any cells that may adhere to the wall to migrate downward to the sample surface.

[0161] In variations of these wall shapes, the wall cross-section may be stepped, e.g. Figure 8 The stepped wall shape can be easier to manufacture than a continuous flared wall while retaining the functional advantages of a flared wall.

[0162] The wall can combine features of these wall shapes. For example, the wall can have an inner surface that flares inward (overhanging above the sample surface) and an outer surface that flares inward (sloping outward from the surrounding base), or vice versa.

[0163] Thus, the wall shape can be optimized for different cell or sample types, as well as different wall and sample surface treatments.

[0164] Figure 9is a plan view of an array of carriers locked on a substrate according to a second embodiment. Depending on the detection application, any number of carriers can be locked on the substrate.

[0165] In other embodiments, surface treatments other than the aforementioned polymer coatings can be applied to the sample surface. For example, the coating can include 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. In addition to applying a coating, the method can include modifying the physical surface of the sample surface of the support to be specifically adapted for receiving adherent cells, such as by modifying a gold cap.

[0166] The final step in the method for manufacturing the carrier system is sterilization (not shown). To sterilize the carrier system, it is placed in sterile packaging, sealed, and then placed in an oven at 120 degrees Celsius for 4 hours. This sterilizes it and produces a ready-to-use sample that can be opened in a sterile cell culture environment.

[0167] Figure 10 This is a micrograph of multiple carriers in a second embodiment used in an adherent cell assay, suspended in a detection solution. During the image capture, the magnetic carriers were aligned facing the camera through appropriate adjustment of the external magnetic field, and the cells were stained to improve 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.

[0168] Figure 10 To place cells on the sample surface of the carrier, the following steps are required. Figure 9 A carrier system, such as a carrier array on a silicon substrate, as shown in FIG, has been placed in a container and immersed in a biocompatible aqueous solution containing adherent cells. When the biocompatible aqueous solution contacts the release layer 214, the release layer dissolves and releases the carriers. If additional time is required to allow the cells to adhere to the carriers while they are on the substrate, an external magnetic field can be applied to keep the carriers in contact with the substrate even after the release layer releases the carriers.

[0169] Once the cells adhere to the carrier, the carrier is released from the substrate and can move freely through the aqueous solution surrounding the carrier system. An external magnetic field can be used to apply a force to the carrier to manipulate and displace the carrier as desired to perform the assay. Figure 10 As shown, a magnetic field can be used to align the carrier for photographic imaging.

Claims

1. A carrier for adhering to a sample for detection, the carrier comprising a sample surface for receiving the adhering sample and a peripheral area surrounding the sample surface and located between the sample surface and an edge of the carrier, wherein the peripheral area comprises a coating for reducing sample adhesion, so that when in use, the adherent sample has weaker adhesion to the peripheral area than to the sample surface.

2. A carrier according to claim 1, wherein during use the carrier can be removably positioned on a substrate for receiving the adherent detection sample, wherein when the carrier is positioned on the substrate, the peripheral area is between the sample surface and the substrate, preferably surrounding the sample surface.

3. A carrier according to claim 2, wherein when the carrier is positioned on the substrate, the height difference between the sample surface and the peripheral area above the substrate is less than 10% of the maximum thickness of the carrier, and preferably less than 1% or 0.1% of the maximum thickness of the carrier.

4. The carrier according to any of the preceding claims, wherein the thickness of the carrier in the region of the sample surface differs from the thickness of the carrier in the peripheral region by less than 10%, and preferably by less than 1% or 0.1%.

5. A carrier according to any one of the preceding claims, wherein the peripheral region is substantially flat and preferably parallel to the substrate when the carrier is positioned on the substrate.

6. A carrier according to any one of the preceding claims, wherein the sample surface is substantially flat and preferably parallel to a substrate when the carrier is positioned on the substrate.

7. The carrier according to any of the preceding claims, wherein the carrier comprises a base portion, and an upper surface of the base portion forms the sample surface and the peripheral area.

8. A carrier according to any preceding claim, comprising a wall between the sample surface and an edge of the carrier.

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

10. The carrier according to claim 8 or 9, wherein at least part of the surface of the wall comprises a coating that reduces adhesion of the sample, so that in use, the adherent sample adheres less strongly to the wall than to the sample surface.

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

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

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

14. The carrier according to any one of claims 8 to 13, wherein at least part of the wall has a height above the sample surface of greater than 1 micron and less than 25 microns.

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

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

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

18. The carrier according to any one of claims 8 to 17, wherein the carrier comprises a base portion, an upper surface of the base portion forming the sample surface, and wherein the wall and the base portion are manufactured using a photolithographic process.

19. The carrier according to any one of claims 8 to 18, wherein the wall comprises a structured photopolymer, preferably a photoresist, SU-8 or AZ-10nXT.

20. The 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 preceding claim, wherein the coating of the peripheral region comprises a non-ionic surfactant.

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

23. The carrier according to any one of the preceding claims, wherein the sample surface comprises a sample adhesion promoting coating.

24. The carrier of claim 23, wherein the sample surface comprises a biofunctionalized coating adapted to receive the adherent sample.

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

26. The carrier of any preceding claim, wherein at least part of the peripheral region has a lateral width or maximum lateral thickness greater than 1 micron and less than 50 microns, or greater than 5 microns and less than 40 microns.

27. The carrier according to any one of the preceding claims, wherein the carrier has a lateral dimension, preferably a smallest lateral dimension, greater than 5 microns and less than 300 microns, or preferably greater than 10 microns and less than 200 microns.

28. A carrier according to any preceding claim, wherein the carrier comprises a magnetic material.

29. A vector system for detection, comprising a plurality of vectors as defined in any one of claims 1 to 28.

30. A method for performing a detection using the vector defined in any one of claims 1 to 28 or using the vector system defined in claim 29, the method comprising the steps of: A plurality of carriers positioned on a substrate are provided, a biocompatible aqueous solution carrying the adherent samples is introduced into the carriers and the substrate, so that the adherent samples adhere to the sample surface of the carriers rather than the peripheral area, and the carriers are released from the substrate.

31. The method for performing detection according to claim 30, wherein the carrier is fixedly positioned on the substrate by a release layer between the carrier and the substrate, and the release layer is activated by the biocompatible aqueous solution to release the carrier.

32. A method for performing detection according to claim 30 or 31, wherein the carrier is magnetic or includes magnetic material and is fixedly positioned on the substrate by an applied magnetic field and is released from the substrate by changing or removing the applied magnetic field.

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