Sample holders

Abstract

A device (2) for holding a sample (50) is provided. The device comprises a first member (6), a second member (12) and a separating member (10) between the first member and the second member. The separating member is formed from a cured separating substance (100). The first member, the second member and the separating member define a sample region (14) for holding a sample.

Description

[0001] Sample holders

[0002] The present invention relates to devices for holding samples such as fluid samples and methods of manufacturing and using such devices.

[0003] Microscopic analysis of fluid samples typically involves imaging a small quantity of the fluid with a microscope, e.g. on a glass slide. For some studies it is desirable to isolate and image very small amounts of the sample, e.g. single molecules macromolecules or nanoparticles. However, isolating such small quantities reliably and consistently can be very difficult.

[0004] Conventional approaches to single-molecule confinement include so-called nanofluidic chips, which feature one or more nanometre-scale channels (nano-channels) though which a fluid sample can flow. The nano-channels are sized to confine individual molecules of the sample, where they can be analysed, e.g. imaged with a microscope. WO 2020 / 104814 A2 discloses a device featuring a network of microfluidic channels with heights of a few hundred nanometres, through which a sample solution is driven.

[0005] However, manufacturing and using conventional nanofluidic chips can be complex and expensive, e.g. requiring clean room facilities and specialist manufacturing equipment.

[0006] An improved approach may be desired.

[0007] According to a first aspect of the present invention there is provided a method of manufacturing a device for holding a sample, the method comprising: depositing a separating substance on a part of a first member; applying a second member to the separating substance; pressing the first member and the second member together to reduce a thickness of the separating substance; and curing the separating substance to form a separating member and to create a sample region for holding a sample defined by the first member, the second member and the separating member.

[0008] According to a second aspect of the present invention there is provided a device for holding a sample comprising: a first member; a second member; and a separating member between the first member and the second member that is formed from a cured separating substance; wherein the first member, the second member and the separating member define a sample region for holding a sample.

[0009] Thus, it will be appreciated by those skilled in the art that, because the sample region is partially defined by the separating member, its height (i.e. a shortest distance from the first member to the second member through the sample region) may be determined by the thickness to which the separating substance is reduced in the pressing step. Thus, through appropriate control of the separating substance and the pressing step, sample regions with desired heights may be consistently and conveniently formed. Moreover, the method can be used to produce extremely thin sample regions, aiding analysis of the sample. In some embodiments, the device is for holding a fluid sample.

[0010] The applicant has recognised that, when performed appropriately, depositing a separating substance and then pressing the members together can produce surprisingly consistent sample region sizes. For instance, capillary forces between the separating substance and the first and second members may mean that a given amount of separating substance adopts a substantially consistent thickness when subject to a wide range of pressing forces. In other words, fine control over the magnitude of the pressing force may not be essential to consistently obtaining a given sample region height. Similarly, a lateral extent to which the separating substance spreads between the first and second members (which may define a lateral dimension of the sample region) may also be substantially consistent when subject to a wide range of pressing forces (although it may be less important to control the lateral extent compared to the thickness).

[0011] Many conventional approaches for forming devices with small sample regions involve subtractive processes, e.g. etching intricate patterns using lithographic techniques. In contrast, it will be appreciated that embodiments of the present invention use an additive manufacturing approach, which may be simpler and more easily scalable. Moreover, it will be recognised that part or all of the method may advantageously be performed using only relatively common and / or inexpensive equipment. Thus, embodiments of the present invention may offer a simpler and / or lower cost way of producing devices for isolating small quantities of samples (e.g. for microscopic analysis) than is currently available. Furthermore, it may be relatively straightforward to automate one or more of the depositing, applying, pressing and curing steps. The sample region’s position between the first and second members may facilitate analysis of the sample. For instance, because the height of the sample region is set by the separating member, the device can be used to prepare a precisely known thickness of a sample. This may facilitate the measurement of sample properties. Moreover, by arranging appropriately the size and shape (e.g. height) of the sample region, particular components of the sample can be isolated in the sample region where they can be sensed (e.g. imaged) separately to the rest of the sample. For instance, the sample region may be sized to admit only those components of a sample under a threshold size (e.g. corresponding to the height of the sample region). In some embodiments, the device may be used to analyse a blood sample, with the sample region having a height selected to exclude some components of the blood samples from the sample region (e.g. red blood cells, platelets, cell bodies) but to allow other components (e.g. extracellular vesicles, nanoparticles) into the sample region where they may be analysed.

[0012] The thickness of the separating member (i.e. corresponding to the thickness of the separating substance after it has been reduced by pressing) may be on the nanometre or near-nanometre scale. This thickness may be defined as a shortest distance from the first member to the second member through the separating member. In some embodiments, a thickness of the separating member is nanoscale or near-nanoscale, e.g. 50 pm or less, 25 pm or less, 10 pm or less, 5 pm or less, 1 pm or less, 500 nm or less, 250 nm or less, 100 nm or less, 50 nm or less, 25 nm or less or even 12 nm or less. The separating member may have a width (e.g. a lateral dimension such as a longest distance through the separating member in a plane perpendicular to the height, or overall) of 100 pm or more, 0.5 mm or more, 1 mm or more or 5 mm or more.

[0013] In a set of embodiments, the sample region has a nanoscale or near-nanoscale height, e.g., a height of 50 pm or less, 25 pm or less, 10 pm or less, 5 pm or less, 1 pm or less, 500 nm or less, 250 nm or less, 100 nm or less, 50 nm or less, 25 nm or less or even 12 nm or less. A thin sample region may enable effectively two-dimensional confinement of a sample. In a set of embodiments, the height of the sample region is based on a size of one or more components of a sample to be analysed (e.g. particles such as cells, molecules, aggregates). The sample region may comprise a height that is less than a size of one or more components of a sample to be excluded from analysis. The sample region may comprise a height that is more than a size of one or more components of a sample to be included for analysis.

[0014] The sample region may have a width (e.g. a lateral dimension such as a diameter in a direction perpendicular to the height) that is larger and preferably much larger than its height. In other words, the sample region may hold the sample in a very thin layer spread over a relatively large area. This may aid analysis. The sample region may have a width (e.g. a longest distance through the sample region in a plane perpendicular to the height, or overall) of 100 pm or more, 0.5 mm or more, 1 mm or more, 5 mm or more or 10 mm or more.

[0015] In a set of embodiments, the sample region is arranged to accommodate only a single layer of a component of the sample. For instance, the sample region may comprise a height that is between one and two times the size of a particle of interest. Sizing the sample region to contain only a single layer of a component of a sample (e.g. a layer that is a single molecule thick) may facilitate improved analysis as explained in more detail below.

[0016] In a set of embodiments, the device comprises a microfluidic or nanofluidic chip.

[0017] In a set of embodiments, the device is arranged to hold a biological sample such as a cell culture. For instance, the components defining the sample region may be biocompatible with one or more biological samples or types of biological samples.

[0018] As explained above, the dimensions of the separating member (and thus of the sample region) may be substantially independent to some variation in the pressing step. However, it may still be possible to control one or more dimensions of the sample region through appropriate selection of parameters of the separating substance itself. In a set of embodiments, the method comprises selecting one or more properties of the separating substance based on a desired thickness and / or lateral extent of the separating member. For instance, the separating substance may be a fluid with a viscosity chosen based on a desired dimension of the separating member.

[0019] Similarly, the method may comprise depositing a quantity of separating substance on the first member based on a desired dimension of the separating member. For instance, the method may comprise depositing a quantity of separating substance of 25 pl or less, 10 pl or less, 5 pl or less, 1 pl or less, 500 nl or less, 100 nl or less, 10 nl or less, or even less than 1 nl, e.g. 500 pl or less, or 300 pl or less. In a set of embodiments, the method comprises using a pipette, a micropipette, a microdispenser (e.g. a piezoelectric-driven picolitre dispenser), a nozzle and / or electrowetting (e.g. using an electrowetting tip) to deposit the separating substance.

[0020] In a set of embodiments, the viscosity and quantity of the separating substance may be selected to achieve a desired separating member thickness when subject to an expected pressing force (or an expected range of pressing forces). In a set of embodiments, the method comprises using a vacuum pick-up tool to apply the second member to the separating substance. In some embodiments, the same tool (e.g. a vacuum pick-up tool) is used to apply the second member to the separating substance and to press the first and second members together.

[0021] Although fine control may not be essential, in some embodiments the method comprises controlling a force with which the first member and the second member are pressed together to obtain a desired thickness of the separating member. In a set of embodiments, the first and second members may be pressed by a human operator. In some embodiments one or more mechanical actuators may be used to press the first and second members together, such as a hydraulic actuator, a piezoelectric actuator and / or an electromagnetic actuator. The use of a mechanical actuator may enable a pressing force to be controlled and repeated reliably.

[0022] The separating member may have an approximately circular shape in a plane perpendicular to its thickness (e.g. in a plane perpendicular to the pressing force). The circular shape may be formed by the separating substance spreading out roughly symmetrically when the first and second members are pressed together). However, symmetry is not essential and even a highly irregular separating member may still effectively form a useful sample region.

[0023] The first member may be approximately circular in a plane perpendicular to its thickness (e.g. in a plane perpendicular to the pressing force). The second member may be approximately circular in a plane perpendicular to its thickness (e.g. in a plane perpendicular to the pressing force). Having one or both members be circular may aid manufacture of a symmetric sample region. However, this is not essential. In some embodiments, the first and / or second member has a non-circular shape in a plane perpendicular to its thickness. For instance, the first and / or second member may have a square or rectangular shape, or any other regular or irregular shape.

[0024] The sample region is defined by the first member, the second member, and the separating member. The sample region may thus comprise space between first and second members that is not occupied by the separating member. The size and shape of the resulting sample region may depend on how the second member is applied to the separating substance. In a set of embodiments, the second member is applied such that the separating substance is located away from an edge of the second member (e.g. at or near the centre of the second member). The second member may be positioned symmetrically about the separating substance. This may result in the separating member being formed as a central (or near central) pillar of a symmetric sample region. For instance, when the second member is circular, the sample region is formed in an annular shape around the separating member. A symmetric sample region may advantageously see fewer fluid currents when it is used to hold a fluid sample.

[0025] Alternatively, the second member may be applied such that the separating substance is located at or near an edge of the second member. This may result in the second member forming a cantilever supported on only one side by the separating member, with the sample region being formed under the cantilever.

[0026] The device may have only one separating member between the first and second members, e.g. formed by depositing the separating substance to only one part of the first member. A single separating member may also be formed from multiple separate depositions of the separating substance which merge into one separating member as the first and second members are pressed together.

[0027] However, in a set of embodiments, the device comprises a plurality of separating members between the first and second members. In such embodiments, the sample region may be defined by the first member, the second member, and some or all of the separating members. The sample region may comprise some or all of the space between first and second members that is not occupied by the separating members. The method may comprise depositing the separating substance on a plurality of different parts of the first member, i.e. to form unconnected portions of separating substance on the first member. The portions may remain unconnected when the first and second members are pressed together. In such embodiments, curing the separating substance may form the plurality of separating members between the first and second members.

[0028] Each portion of separating substance may comprise a quantity of 25 pl or less, 10 pl or less, 5 pl or less, 1 pl or less, 500 nl or less, 100 nl or less, 10 nl or less, or even less than 1 nl, e.g. 500 pl or less, or 300 pl or less.

[0029] Using multiple separating members may provide the device with increased mechanical stability and / or finer control over the sample region shape. For instance, the shape and position of the separating members may be selected to provide a sample region with a desired shape between the separating members (e.g. a narrow channel between the separating members). In a set of embodiments the sample region comprises a shortest distance between a pair of separating members of 1 mm or less, 500 pm or less, 200 pm or less, 50 pm or less, 20 pm or less or 1 pm or less. In a set of embodiments, the device comprises three or more separating members. The use of three separating members may provide a very stable structure between the first and second members. The separating members may be spaced evenly (e.g. with a similar or identical spacing between multiple pairs of separating members).

[0030] As explained above, a separating member may be formed from a single deposition of the separating substance, or from multiple separate depositions that merge together during the pressing. Using multiple separate depositions of the separating substance can facilitate the manufacture of more complex separating member shapes and / or sample region shapes (e.g. asymmetric shapes), e.g. by multiple depositions merging together and / or by multiple depositions forming multiple separating members. In some embodiments, the method comprises multiple separate depositions of the separating substance, with the volume and / or position of the depositions being controlled to produce one or more separating members defining a sample region with a desired shape. With appropriate control over multiple depositions of the separating substance, complex sample region geometries may be obtained.

[0031] In some embodiments, the device comprises only one sample region, defined by the first member, the second member, and the separating member. However, it sometimes be useful to have provide several separate sample regions with the same device. In a set of embodiments, the device comprises a plurality of sample regions.

[0032] The plurality of sample regions may be defined by distinct sets of first, second and separating members. However, in some embodiments, the first member, the second member and / or the separating member partially define multiple sample regions. In other words, at least one of the first, second and separating members may be shared between multiple sample regions. For instance, the separating substance may be deposited on a plurality of different parts of a single first member, and a corresponding plurality of second members may then be applied to form a plurality of distinct sample regions. Conversely, the separating substance may be deposited on a plurality of different first members, and a single second member applied to all of these to form a plurality of distinct sample regions.

[0033] In some embodiments featuring multiple sample regions, forming the plurality of sample regions comprises performing at least one of the depositing, applying, pressing and curing steps for at least two sample regions at the same time (i.e. performing one of these steps for multiple sample regions in parallel). For instance, a light (e.g. UV) curing step may be performed for two or more sample regions in parallel by exposing distinct portions of the separating substance to the same light source. Additionally or alternatively, depositing a plurality of portions of separating substance may be performed for two or more sample regions in parallel by a corresponding plurality of pipetting machines driven in parallel.

[0034] The separating substance may comprise any suitable substance known in the art perse (i.e. that can be deposited sufficiently accurately, whose thickness can be reduced by pressing, and that is curable to form the separating member). In a set of embodiments, the separating substance is a liquid. The separating substance may comprise a mixture of two or more components. The separating member may be transparent to one or more wavelengths of light, e.g. to aid optical analysis of the sample. The separating substance may also be transparent. In a set of embodiments, the separating substance is biocompatible with one or more biological samples or types of biological samples.

[0035] Curing the separating substance may comprise any suitable curing mechanism known in the art perse. Curing may comprise simply waiting for a separating substance (e.g. a single-part glue) to dry. Curing the separating substance may comprise inducing crosslinking in the separating substance and / or inducing polymerisation in the separating substance. In a set of embodiments, the separating substance is light-curable, e.g. a negative photoresist. In other words, curing the separating substance may comprise exposing the separating substance to one or more wavelengths of light (e.g. UV light). In a set of embodiments, the separating substance is a UV- curable negative photoresist (e.g.mr-UVCur26SF offered by micro resist technology GmbH). The use of light to cure the separating substance may be particularly convenient because, as explained below, one or both of the first and second members may advantageously be transparent to aid analysis of the contents of the resulting sample region. Moreover, light-curing techniques may be relatively quick and simple to implement. For instance, many factories / laboratories already feature or have access to UV sterilisation equipment that may easily be repurposed for curing.

[0036] In some embodiments, additionally or alternatively, other curing mechanisms may be used. For instance, curing the separating substance may comprise heating or cooling the separating substance. In some embodiments, the separating substance comprises a multi-component substance arranged to cure by a reaction between multiple mixed components. Such embodiments may comprise mixing the components together to form the separating substance before depositing the separating substance on the first member. The curing step may then simply require waiting for the reaction between the pre-mixed components to complete and / or triggering or accelerating the reaction by applying one or more stimuli (e.g. heat, light). Additionally or alternatively, the method may comprise adding one or more curing components to the separating substance after it has been deposited on the first member to cause it to cure. In some embodiments, a combination of curing mechanisms is used to cure the separating substance (e.g. light treatment and then heat treatment).

[0037] In a set of embodiments, the first member is transparent to one or more wavelengths of light, to aid use of the device for analysing a sample in the sample region. In some embodiments, the first member comprises one or more of the following materials: glass, plastic, crystal (e.g. CaF, SiN, FS), silicon, SiN, fused silica.

[0038] In a set of embodiments, additionally or alternatively, the second member is transparent to one or more wavelengths of light, to aid use of the device for analysing a sample in the sample region. In some embodiments, the second member comprises one or more of the following materials: glass, plastic, crystal (e.g. CaF, SiN, FS), silicon, SiN, fused silica. The first and second members may be formed from the same material, but this is not essential.

[0039] The first and / or second member may comprise a microscope slide, the bottom of a glass bottom dish or a petri dish. The first and / or second member may comprise a cover slip. The first and / or second member may comprise a silicon wafer. The first and / or second member may comprise a thickness of 300 pm or less, 200 pm or less, 150 pm or less, 100 pm or less, 50 pm or less or 10 pm or less.

[0040] In some embodiments, the first member comprises a first surface that partially defines the sample region, and the second member comprises a second surface that partially defines the sample region. For instance, the first and second surfaces may define upper and lower boundaries of the sample region (or vice-versa). The first surface may be substantially planar. Additionally or alternatively, the second surface may be substantially planar. The first and / or second members may themselves be substantially planar (e.g. comprising a cover slip with two flat parallel surfaces). In a set of embodiments, the first and second planar surfaces are parallel, such that the sample region has a uniform height between the first and second surfaces. Alternatively, the first and second planar surfaces may be inclined relative to each other. A gradual change in height over the sample region may be desirable for some analyses.

[0041] The first and second surfaces are preferably featureless, e.g. completely flat. Using featureless surfaces to form the sample region may simplify manufacture, e.g. as precise lateral alignment of surface features may not be required. However, in some embodiments, one or both of the first and second surfaces comprises one or more raised or inset surface features. For instance, the first and / or second surface may comprise one or more carvings, microstructures, electrodes, channels and / or biofilms. The first and / or second surface may comprise one or more pockets (e.g. nanometre-scale pockets) arranged to hold particles (e.g. molecules) of interest in a sample, e.g. to increase their residence time in the sample region. The method may comprise forming one or more features in or on the first and / or second surface. These may be formed prior to assembly (e.g. etching microstructures into the first or second member before they are otherwise used), or during or after the device is otherwise assembled (e.g. adding a biological substance to the sample region to form a biofilm on the first and / or second surface).

[0042] The one or more surface features may help to guide or confine a sample (or particular components of a sample) to particular areas of the sample region. For instance, one or more surface features may be used to block or permit different components of a sample reaching areas of the sample region based on size (e.g. to exclude cell bodies, giant vesicles, nanoparticles, etc.).

[0043] One or more of the surface features may be formed using lithography.

[0044] In a set of embodiments, one or both one or both of the first and second surfaces is coated (i.e. the device comprises a coating on one or both of the first and second surfaces). For instance, one or both of the first and second surfaces may be coated with a lipid bilayers. One or both of the first and second surfaces may be biofunctionalized against protein adhesion.

[0045] In a set of embodiments, the first and / or second surface comprises a Raman-enhancing nanostructure. For instance, the first and / or second surface may comprise one or more metal nanopillars, periodic metal dot arrays or ellipsoids, bow-tie structures and / or plasmonic structures. One or more of these may be produced by chemical etching. In some embodiments, the first and / or second surface comprises a metal (e.g. gold) coating for Raman enhancement.

[0046] In a set of embodiments, the first and / or second surface is reflective to one or more wavelengths of light. This may be helpful for increasing contrast in scattering-based imaging of the sample. One or both surfaces may feature a reflective coating, and / or the material from which the first and / or second surface is made may be inherently reflective (e.g. a naturally reflective silicon wafer) and / or the first and / or second surface may be treated to become reflective.

[0047] In a set of embodiments, the first and / or second member comprises one or more electrodes for applying an electric field to the sample region (e.g. located on the first and / or second surface). For instance, the device may comprise a pair of electrodes positioned on opposite sides of the sample region. This may facilitate electrophoretic measurements of a sample in the sample region.

[0048] In a set of embodiments, the device comprises a well comprising a floor and at least one wall extending at least partially perpendicular to the floor and arranged to constrain a sample within the well. The well may be a fluid well arranged to constrain a fluid sample. The device may comprise a standard microscopy sample well.

[0049] In some embodiments, the floor of the well comprises the first member (on which the separating substance is deposited). In such embodiments, the second member (or at least the second surface) may be located inside the well, e.g. located partially or completely below an upper extent of the wall(s) of the well. In some such embodiments, the second member is positioned centrally within the well. Alternatively, the floor of the well may comprise the second member and the first member (or the first surface) may be located inside the well. In such embodiments, the first member may be positioned centrally within the well.

[0050] In other words, in some embodiments where the device comprises a well, the member to which the separating substance is applied can either be the floor of the well or a member that is located inside the well and spaced from the floor by the separating substance. In such embodiments, the member that is located inside the well and spaced from the floor by the separating substance acts as an upper floor of a double-floor chamber (with the floor of the well acting as the lower floor and the sample region existing between the floors).

[0051] The well may comprise a single continuous wall, i.e. without any abrupt changes in direction. For instance, the well may comprise a single circular or elliptical wall. Use of a continuous wall may mitigate the formation of undesirable fluid currents in the well. However, alternatively, the well may comprise a plurality of walls. The walls may be straight or curved. For instance, the well may have a semi-circular shape, a square shape, a rectangular shape, or any other irregular or regular shape. A first or second member that is located inside the well may have a corresponding shape to the shape of the wall. For instance, a circular member may be located inside a circular-walled well.

[0052] The device comprising a fluid well may facilitate its use for holding and analysing fluid samples, because the well can constrain the fluid sample and encourage the fluid sample into the sample region. Because the wall of the well constrains the fluid sample, it may also mitigate fluid currents around the sample region. Preferably, the sample region extends close to the wall of the fluid well (i.e. the upper floor extends close to the wall of the fluid well), e.g. to within 5 mm or less, 1 mm or less, or 0.5 mm or less.

[0053] In a set of embodiments, the sample region is symmetric within the well (i.e. the upper floor is symmetric within the well). For instance, the sample region may have an annular shape that is centred within a circular well. This may further reduce fluid currents during use.

[0054] As mentioned above, the device may comprise a plurality of sample regions. In a set of embodiments, the device comprises a plurality of wells, each containing a sample region defined by first, second and separating members. For instance, the device may comprise a multi-well plate. The multi-well plate itself may be considered as a common first or second member.

[0055] The device may be arranged with an intended orientation for use. For instance, in embodiments where the device comprises a well, the operational orientation may be that in which a sample is held in the well by gravity. The operational orientation may be when the second member is positioned directly above the first member, or vice versa (e.g. with both oriented horizontally). In other words, the first member may be at the top of the device when it is used, or the second member may be at the top of the device when it is used. When viewed another way, the separating substance may be deposited on what will form a top member of the device, or on what will form a bottom member of the device.

[0056] After the device is physically fabricated, it may be useful to measure one or more features of the device, e.g. for record-keeping, quality control and / or to provide feedback for refining one or more of the manufacturing steps. In a set of embodiments, the method comprises measuring a position and / or shape and / or size of the separating member (e.g. for adjusting the depositing step), measuring a position of the second member (e.g. for adjusting the applying and / or pressing step) and / or measuring a height of the sample region (e.g. for adjusting a quantity or viscosity of separating substance used and / or for adjusting the pressing step). Performing one or more of these measurements may comprise using interference microscopy, phase-contrast microscopy or sequential laser focusing.

[0057] When viewed from a third aspect, the invention extends to a method of analysing a sample using the device disclosed herein, the method comprising: introducing a sample to the sample region of the device; and sensing the sample. The device may be useful for analysing a wide variety of samples. In some embodiments, the sample is a fluid sample. The sample may consist of a liquid, e.g. comprising only molecules in a liquid phase. However, many real-world fluid samples comprise a mixture of a liquid with other particles, e.g. in suspension or solution. The sample may contain a mixture of multiple different substances in different phases. The sample may comprise a biological sample, e.g., comprising water, cells, proteins, DNA, mineral ions, dissolved gases and / or other biological structures or substances. In some embodiments the sample may contain functional DNA structures (e.g. DNA origami constructs). In some such embodiments the sample also contains a target of said structures (e.g. for viruses, proteins, biomacromolecules).

[0058] In some embodiments the sample may be introduced as a fluid, but not still be a fluid when it is sensed. For instance the method may comprise introducing a fluid sample and allowing the fluid sample to dry to form a film in the sample region which is then sensed. For instance, a biological sample may be dried to bio film layer in which biological structures are immobilised, which may aid sensing.

[0059] The sample region may be empty when the sample is introduced. However, some embodiments comprise at least partially filling the sample region with a sample medium prior to prior to introducing the sample. For instance, the sample region may be filled with a hydrogel, e.g. to facilitate single molecule electrophoresis. A set of embodiments comprises filling the sample region at least partially with water and then adding agarose to form an ultra-thin layer of agarose hydrogel in the sample region.

[0060] In some embodiments, one or more additives for facilitating sensing are added to the sample (e.g. nanoparticles with functionalised surfaces). These may be added to the sample region before the sample is introduced, after the sample is introduced, or they may be mixed into the sample separately beforehand. For instance, one or more constituents of the sample may react in a particular way with the additives and induce a reaction that can be sensed (e.g. a catalytic surface reaction on the functional nanoparticles to make them spin, propagate or produce a colorimetric readout signal).

[0061] In a set of embodiments, the sample comprises a first component having a first particle size (e.g. diameter), and a second component having a second, larger particle size. For instance, the sample may be a blood sample comprising extracellular vesicles having a size of roughly 100 nm, and red blood cells having a size of roughly 5 pm. In some embodiments, the sample region is arranged to exclude the second component (e.g. red blood cells). The sample region may be arranged to admit the first component (e.g. extracellular vesicles). For instance, the sample region may have a height (defined by the thickness of the separating member) that is greater than the first size and less than the second size.

[0062] The ability to selectively admit components of a sample into the sample region for sensing may allows existing sensing techniques (e.g. optical microscopes) to be combined with nanofluidic particle analysis. This may facilitate the study of protein / protein interactions, nanoscale particles emitted by cell cultures, and extracellular proteins, e.g., using only conventional optical microscopy.

[0063] As explained above, in a set of embodiments, the sample region is arranged to accommodate only a single layer of a component of the sample. For instance the sample region may accommodate a layer of a first component in the sample that is one particle (e.g. one extracellular vesicle) thick. Isolating single particles may improve the quality of sensing, e.g. enabling the device to be used for single-molecule detection.

[0064] In some embodiments, the sample is sensed through the first and / or second member. In a set of embodiments, the sample is sensed using light passing through the first and / or second member. Additionally or alternatively, the sample may be sensed from another direction, e.g. from a side of the device. In such embodiments the sample may be sensed using light that has not passed through either of the first and / or second members.

[0065] Light used to sense the sample may be in the visible spectrum, or it may be non-visible (e.g. UV, DUV, X-ray, IR or Mid-IR). In a set of embodiments, an optical microscope is used to sense the sample. The method may comprise transmission microscopy, i.e. comprising directing light into the sample region through one of the first and second members, and detecting light from the sample region through the other of the first and second members. The method may comprise dark field microscopy and / or widefield microscopy. The detected light may be scattered light and / or fluorescence from the sample. The microscope may be operated as an inverted microscope, i.e. with the light source located physically above the sample holder and the sensing optics below the sample holder.

[0066] The device may be arranged such that the light that passes through the first and / or second member for sensing the sample does not pass through any of the sample that is not in the sample region. For instance, where the sample region is part of a larger sample-holding volume such as a well, the larger volume may hold the rest of the sample out of a line-of-sight of the sample region. However, this is not essential and in other embodiments the light that passes through the first and / or second member for sensing the sample may also pass through parts of the sample that are not in the sample region. In such embodiments it will be recognised that the first and second member still provide spatial separation between the portion of the sample in the sample region and other parts of the sample and thus enable the sample region to be sensed separately, e.g. via appropriate focusing of the light and / or other optical sectioning techniques.

[0067] In a set of embodiments, sensing the sample comprises imaging the sample (e.g. using microscopy). Additionally or alternatively, sensing the sample may comprise obtaining one or more spectra from the sample (e.g. using spectroscopy such as Raman spectroscopy).

[0068] The sample region may be part of a larger sample-holding volume, e.g. a fluid well. Introducing a sample to the sample region may comprise introducing the sample to the larger sampleholding volume and allowing it to move into the sample region. This may allow a user to work with relatively large amounts of sample (e.g. 100s pl), even though only very small amounts may end up in the sample region for sensing. In other words, the sample region may enable a very small quantity of the sample to be isolated from the rest of the sample.

[0069] In some embodiments, the method comprises applying one or more stimuli to the sample. For instance, electrodes may be used to apply a voltage to the sample before or as it is sensed.

[0070] In a set of embodiments, the method comprises performing one or more of: molecular binding assays, single molecule detection and cell culture analysis. For, instance, embodiments may comprise analysing cell cultures such as Alzheimer’s, Parkinson’s or Cancer models to determine information about extracellular space, such as amyloid and aggregate sizing. Embodiments may also be used for Antibody-protein binding testing (Dissociation studies), nanoparticle sizing and binding, exosome sizing in cell cultures, analysing protein-protein interactions, analysing liquid / liquid phase transitions, mRNA binding assays, molecular sizing, single molecule FRET or label-free specimen detection (e.g. using Interferometric scattering microscopy or dark field microscopy).

[0071] In a set of embodiments, sensing the sample comprises tracking one or more components of the sample in the sample region. For instance, the movement and / or rotation of particles in the sample may be tracked over time. The confinement provided by a potentially very thin sample region may facilitate this tracking.

[0072] Features of any aspect or embodiment described herein may, wherever appropriate, be applied to any other aspect or embodiment described herein. Where reference is made to different embodiments, it should be understood that these are not necessarily distinct but may overlap. It will be appreciated that all of the structural and functional features of the device described above with reference to the method of manufacturing according to the first aspect may also apply to the other aspects of the invention, and vice versa.

[0073] One or more non-limiting examples will now be described, by way of example only, and with reference to the accompanying figures in which:

[0074] Figure 1 is a schematic diagram of a device for holding a sample according to an embodiment of the present invention;

[0075] Figure 2 is a schematic cross-section diagram of the device of Figure 1 , in a plane marked by the line labelled F2;

[0076] Figure 3 is a schematic diagram illustrating the use of the device for holding and analysing a sample;

[0077] Figures 4-8 are schematic diagrams showing various steps in a method of manufacturing the device shown in Figures 1 , 2 and 3;

[0078] Figure 9 is a schematic diagram of a device for holding a sample according to another embodiment of the present invention.

[0079] Figure 10 is a schematic cross-section diagram of the device of Figure 9, in a plane marked by the line labelled F10; and

[0080] Figure 11 is a schematic diagram of a device for holding a sample according to another embodiment of the present invention.

[0081] Figure 12 is a schematic cross-section diagram of the device of Figure 11 , in a plane marked by the line labelled F12; and

[0082] Figure 13 is a schematic diagram of a multi-well plate comprising multiple such devices.

[0083] Figures 1 and 2 show a device 2 for holding a sample such as a fluid sample. The device 2 comprises a well 4 (e.g. of a multi-well plate) comprising a floor 6 and a circular wall 8 that extends from the floor 6. The well 4 is made of a transparent material such as plastic, glass, crystal, silicon, SiN or fused silica. The well 4 is approximately 10 mm in diameter and approximately 10 mm deep (Figures 1 and 2 are not drawn to scale). In other examples, other sizes of well may be used.

[0084] A separating member 10 is located in the centre of the well 4. The separating member 10 is formed from cured UV negative photoresist.

[0085] A cover slip 12 is positioned on top of the separating member 10. The cover slip 12 is made from a transparent material such as plastic, glass, crystal, silicon, SiN or fused silica. The cover slip 12 is approximately 150 pm thick and approximately 8 mm in diameter (leaving a ~1 mm gap between the edge of the cover slip 12 and the wall 8). In other examples, other sizes of cover slip may be used.

[0086] The diameter of the cover slip 12 is larger than that of the separating member 10, i.e. the cover slip 12 extends past the edge of the separating member 10. A sample region 14 is thus defined by the floor 6 of the well 4, the separating member 10 and the cover slip 12, extending to the radially-outer edge of the cover slip 12. The sample region 14 has annular cylindrical shape.

[0087] The sample region 14 is open at the side opposite to the separating member 10 (i.e. the radially outer edge of the sample region 14). This allows a sample 50 to be introduced to the sample region 14 (as illustrated in Figure 3).

[0088] The cover slip 12 and the floor 6 are both planar. The cover slip 12 extends parallel to the floor 4. The separating member 10 has a constant thickness. As a result, the sample region 14 has a uniform height H equal to the thickness of the separating member 10. In this example, the separating member 10 (and the sample region 14) is approximately 100 nm thick. In other examples, other thicknesses of separating member 10 may be used (e.g. ranging between 12nm and 50 pm thick).

[0089] Figure 3 shows use of the device 2 for analysing a fluid sample (e.g. by microscopy). A fluid sample 50 is added to the well 4, which flows around and under the cover slip 12 to fill the sample region 14. The fluid sample 50 contains a first particle component 51 and a second particle component 53. The second particle component 53 is much larger than the first particle component 51.

[0090] The height H of the sample region 14 is selected to admit the first particles 51 and exclude the second particles 53. Moreover, the height is sufficient small that the sample region 14 contains only a single layer of the first particles 51.

[0091] A user 52 (e.g. using a microscope) can thus observe a single layer of the first particles 51 through the cover slip 12. The user 52 may image the first particles 51 or perform other analysis (e.g. spectroscopy). In some examples, the fluid sample 50 may fill the well 4 to above the top of the cover slip 12. In such examples a user may use optical sectioning techniques to image the thin layer of the first particles 51 isolated in the sample region 14. In Figure 3 the user 52 is shown observing the sample from above (solid line) or below (dashed line), but it will be recognised that the device 2 may facilitate analysis from any direction or combination of directions, including from the side(s) of the device 2.

[0092] A method of manufacturing the device 2 will now be described with additional reference to Figures 4-8.

[0093] The method begins with an empty well 4 as shown in Figure 4. The well 4 may be a standard microscopy sample well, e.g. one well of a multi-well plate.

[0094] In a first step, shown in Figure 5, a small quantity of a liquid UV-curable negative photoresist 100 (e.g. mr-UVCur26SF) is deposited in the centre of the floor 6 of the well 4 using a pipette 102. The quantity of the deposited liquid photoresist 100 is precisely controlled using the pipette 102.

[0095] In a second step, shown in Figure 6, the cover slip 12 is applied to the top of the liquid photoresist 100 using a vacuum pick-up tool 104.

[0096] In a third step, shown in Figure 7, downward pressure is applied to the cover slip 12 to press it toward the floor 6 of the well 4. This squeezes the liquid photoresist 100 outwards and reduces its thickness. The downward force is applied via the vacuum pick-up tool 104 using a mechanical actuator 105.

[0097] Next, shown in Figure 8, the whole assembly is exposed to UV light 106 from a UV light source 107. This cures the liquid photoresist 100 to form the separating member 10. A desired thickness of the separating member 10 (and the sample region 14) is obtained by suitable selection of the type of photoresist 100 used, the quantity of photoresist 100 deposited by the pipette 102 and the magnitude of downward force applied by the actuator 105.

[0098] Figures 9 and 10 show another device 202 for holding a sample. Much of the structure of the device 202 is the same as the device 2 described above. The device 202 comprises a well 204 comprising a floor 206 and a circular wall 208 that extends from the floor 206. The well 204 is made of a transparent material.

[0099] However, in contrast to the device 2 described above the device 202 comprises three separating members 210A, 210B, 201C. The separating members 210A, 210B, 201C are formed from cured UV negative photoresist. The separating members 210A, 210B, 201C are substantially identical and are spaced evenly towards the middle of the well 204. A cover slip 212 is positioned on top of the separating members 210A, 210B, 201C. The cover slip 212 is made from a transparent material.

[0100] The cover slip 212 extends past the radially-outer edges of the separating member 210A, 210B, 201 C. A sample region 214 is defined by the floor 206 of the well 204, the separating members 210A, 210B, 201C and the cover slip 212, extending to the radially-outer edge of the cover slip 212.

[0101] The sample region 214 is open at the edge of the cover slip 212, allowing a sample to be introduced to the sample region 214.

[0102] The cover slip 212 and the floor 206 are both planar. The cover slip 212 extends parallel to the floor 204. The separating members 210A, 210B, 210C have equal constant thicknesses. As a result, the sample region 214 has a uniform height equal to the thicknesses of the separating members 210A, 210B, 210C. In this example, the separating members 210A, 210B, 210C (and the sample region 214) are approximately 100 nm thick. In other examples, other thicknesses of separating members 210A, 210B, 210C may be used (e.g. ranging between 12nm and 50 pm thick).

[0103] The device 202 may be used to hold a sample for analysis as described above with reference to Figure 3. Using three evenly spaced separating members 210A, 210B, 210C can provide increased mechanical stability and control over the sample region shape.

[0104] Figure 13 shows a device 302 according to another embodiment of the invention. The device 302 is a multi-well plate comprising 24 identical wells 304. Each of the wells 304 has the same structure as the device 2 described above and illustrated in Figures 1-8. The multi-well plate 302 thus provides multiple sample regions for holding and analysing samples. Providing multiple sample regions on the same multi-wave plate 302 may facilitate efficient parallel analyses. In this embodiment the sample regions are the same, but this is not essential and in some embodiments a multi-well plate may comprise various different structures forming different sample regions.

[0105] While the invention has been described in detail in connection with only a limited number of embodiments, it should be readily understood that the invention is not limited to such disclosed embodiments. Rather, the invention can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the scope of the invention. Additionally, while various embodiments of the invention have been described, it is to be understood that aspects of the invention may include only some of the described embodiments. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.

Claims

Claims1. A method of manufacturing a device for holding a sample, the method comprising: depositing a separating substance on a part of a first member; applying a second member to the separating substance; pressing the first member and the second member together to reduce a thickness of the separating substance; and curing the separating substance to form a separating member and to create a sample region for holding a sample defined by the first member, the second member and the separating member.

2. The method of claim 1 , wherein the sample region has a height of 5 pm or less.

3. The method of claim 1 or 2, comprising selecting one or more properties of the separating substance based on a desired thickness and / or lateral extent of the separating member.

4. The method of any preceding claim, comprising depositing a quantity of separating substance on the first member based on a desired dimension of the separating member.

5. The method of any preceding claim, comprising controlling a force with which the first member and the second member are pressed together to obtain a desired thickness of the separating member.

6. The method of any preceding claim, comprising positioning the second member symmetrically about the separating substance.

7. The method of any preceding claim, comprising depositing the separating substance on a plurality of different parts of the first member to form unconnected portions of separating substance on the first member, the portions remaining unconnected when the first and second members are pressed together, such that curing the separating substance forms a plurality of separating members between the first and second members.

8. The method of claim 7, wherein the sample region comprises a shortest distance between a pair of separating members of 1 mm or less.

9. The method of any preceding claim, wherein curing the separating substance comprises exposing the separating substance to one or more wavelengths of light.

10. The method of any preceding claim, wherein the first member and / or the second member is transparent to one or more wavelengths of light.

11. The method of any preceding claim, wherein the first member comprises a first surface that is substantially planar and partially defines the sample region, and the second member comprises a second surface that is substantially planar and partially defines the sample region.

12. The method of claim 11 , wherein the first and second surfaces are parallel, such that the sample region has a uniform height between the first and second surfaces.

13. The method of claim 11 or 12, wherein one or both of the first and second surfaces comprises one or more raised or inset surface features14. The method of any preceding claim, wherein the device comprises a well comprising a floor and at least one wall extending at least partially perpendicular to the floor and arranged to constrain within the well, wherein the floor of the well comprises the first member and the second member is located inside the well.

15. The method of claim 14, wherein the sample region is symmetric within the well.

16. The method of claim 14 or 15, wherein the device comprises a plurality of wells, each containing a sample region defined by first, second and separating members.

17. A device for holding a sample comprising: a first member; a second member; and a separating member between the first member and the second member that is formed from a cured separating substance; wherein the first member, the second member and the separating member define a sample region for holding a sample.

18. The device of claim 17, wherein the separating member comprises a central pillar of a symmetric sample region.

19. The device of claim 17 or 18, wherein the sample region has a height of 1 pm or less and a width of 1 mm or more.

20. The device of any of claims 17-19, comprising a fluid well comprising a floor and at least one wall extending at least partially perpendicular to the floor and arranged to constrain fluid within the well, wherein the floor of the fluid well comprises the first member and the second member is located inside the well.

21. A method of analysing a sample using the device of any of claims 17-20, the method comprising: introducing a sample to the sample region of the device; and sensing the sample.

22. The method of claim 21 , wherein the sample comprises a biological sample.

23. The method of claim 21 or 22, wherein the sample comprises a first component having a first particle size and a second component having a second, larger particle size, and the sample region is arranged to exclude the second component and admit the first component.

24. The method of any of claims 21-23, comprising sensing the sample using light passing through the first and / or second member.

25. The method of any of claims 21-24, wherein the device comprises a fluid well and introducing the fluid sample to the sample region comprises introducing the sample to the fluid well and allowing it to move into the sample region.

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