Patent Grant
12303887
The present application is a U.S. National Stage of International Application No. PCT/GB2017/051065 filed on Apr. 18, 2017, which claims the benefit of U.K. Patent Application Nos. 1614139.2, 1614146.7, 1614150.9, 1614153.3, 1614157.4 all filed on Aug. 18, 2016 and International Application No. PCT/GB2016/053204 filed on Oct. 14, 2016, the entire disclosures of all of which are incorporated herein by reference in their entireties.
The invention relates to creating a microfluidic arrangement by dividing a body of a first liquid into a plurality of sub-bodies of liquid that are separated from each other by a second liquid and held stably by surface tension. The sub-bodies can be used to provide isolated samples containing material to be investigated, such as living cells or other biological material.
Microwell plates are widely used for studies involving biological material. Miniaturisation of the wells allows large numbers of wells to be provided in the same plate. For example, plates having more than 1000 wells, each having a volume in the region of tens of nanolitres, are known. Further miniaturisation is difficult, however, due to the intrinsic need to provide solid walls that separate the wells from each other. The thickness of these walls reduces the surface area available for the wells. For a typical plate having 1536 wells, for example, the walls would be expected to occupy about 60% of the available surface for current designs. For higher densities the proportion of the surface area made unavailable by the walls will increase further.
A further obstacle to miniaturisation of microwell plates is the difficulty of adding liquids to small wells defined by physical walls. For liquid to be added reliably to a well (i.e. in a way which avoids trapping of air beneath the liquid), a tip needs to be advanced accurately to the bottom of the well without the tip or any liquid attached to the tip touching the walls of the well. If contact is made with the walls before the liquid reaches the bottom of the well it is likely that a meniscus will form with the wall and trap air beneath the liquid. This may mean that liquid cannot reach the bottom of the well.
Microwell plates also lack flexibility because the size of the wells and the number of wells per plate is fixed. Furthermore, biological and chemical compatibility can be limited by the need to use a material that can form the structures corresponding to the wells in an efficient manner. For example, for high density plates it may be necessary to use a material such as polydimethylsiloxane (PDMS), but untreated PDMS has poor biological and chemical compatibility because it teaches toxin and reacts with organic solvents.
EP 1 527 888 A2 discloses an alternative approach in which ink jet printing is used to form an array of closely spaced droplets of growth medium for culture and analysis of biological material. This approach provides more flexibility than a traditional microwell plate but requires sophisticated equipment to perform the printing. Additionally, it is time consuming to add further material to the droplets after the droplets have been formed and there is significant footprint not wetted by the resultant sessile drops as they do not tessellate.
It is an object of the invention to provide an alternative way of creating a microfluidic arrangement that at least partially addresses one or more of the challenges discussed above.
According to an aspect of the invention, there is provided a method of manufacturing a microfluidic arrangement, comprising: providing a continuous body of a first liquid in direct contact with a substrate; providing a second liquid in direct contact with the first liquid and covering the first liquid, such that the first liquid is in direct contact exclusively with the second liquid and the substrate; and forcing the second liquid through the first liquid and into contact with the substrate in selected regions of the substrate in order to divide the continuous body of the first liquid into a plurality of sub-bodies of the first liquid that are separated from each other by the second liquid, wherein: the first liquid is immiscible with the second liquid; and surface tension stably holds the plurality of sub-bodies of the first liquid separated from each other by the second liquid.
The method allows sub-bodies of a liquid to be formed flexibly on a substrate without any mechanical or chemical structures being provided beforehand to define the geometry of the sub-bodies. The shapes and sizes of the sub-bodies are defined by the shapes and sizes of the selected regions of the substrate that the second liquid is forced to contact. As described below, the choice of the selected regions is relatively unrestricted. It is possible to create extremely small sub-bodies, for example of the order of 100 microns or smaller, which would be difficult or impossible to create at reasonable cost using standard microwell plate manufacturing techniques. The sub-bodies can also be positioned much closer to each other than is possible using microwell plates with physical walls. The liquid walls of embodiments of the present disclosure typically have a thickness of 70-120 microns, which allows more than 90% of the surface area of the microfluidic arrangement to be available for containing liquids to be manipulated. Furthermore, there are no solid walls to interfere with adding further liquid to any of the sub-bodies.
In comparison with arrays of droplets deposited by ink jet printing or the like, the method avoids the need for sophisticated printing equipment and can achieve higher space filling efficiency (because the shapes of the sub-bodies do not need to be circular). Materials to be investigated (e.g. cells) and test substances (e.g. drugs) can be added to multiple sub-bodies simultaneously by adding them to the continuous body of the first liquid before it is divided into the sub-bodies. Concentration gradients can be imposed in strips of the first liquid and the strips can be divided into sub-bodies to quickly and easily create multiple samples containing different concentrations of components. The inventors have furthermore found that depositing fluid into the sub-bodies after they have been formed can be achieved more efficiently (merging occurs more quickly) for sub-bodies that do not have a round footprint (e.g. substantially square or rectangular sub-bodies). Without wishing to be bound by theory, it is thought this effect may be influenced by the reduced symmetry of the non-circular sub-bodies and/or by the fact that they can be flatter. Non-circular sub-bodies can be formed easily using methods of the disclosure.
In an embodiment, the forcing of the second liquid through the first liquid is performed by moving a distal tip of a separator member through the first liquid over the selected regions of the substrate; and at least a portion of the distal tip of the separator member has a surface energy density that is lower in respect of contact with the second liquid than in respect of contact with the first liquid.
The surface properties of the separator member allow the second liquid to be dragged through the first liquid quickly and efficiently, allowing the dividing process to be performed reliably and at high speed. The simple approach of moving a separator element through the first liquid can be implemented using relatively inexpensive hardware.
In an embodiment, the continuous body of the first liquid is laterally constrained predominantly by surface tension.
Forming the continuous body in this way is desirable because it means that the first liquid does not have to spread out over the whole surface of the receptacle. This means that the shape can be controlled independently of the shape of the receptacle, which allows more optimal space filling. The continuous body can be arranged to be square or rectangular, for example, which allows an array of square or rectangular sub-bodies to be formed with minimal wastage of the first liquid, even when the receptacle itself is not square or rectangular. Furthermore, the clearance between the continuous body and the lateral walls of the receptacle can reduce the risk of interference between the walls and any elements being used to form the continuous body or to divide the continuous body into sub-bodies. Multiple discrete continuous bodies (e.g. squares or rectangles) can be formed in this way. The inventors have furthermore found that the depth of the first liquid can be higher, without the thickness of the layer being disrupted by the denser second liquid above, when the first liquid is laterally constrained predominantly by surface tension rather than by lateral walls of a receptacle.
In an embodiment, the forcing of the second liquid through the first liquid comprises the following steps in order: dividing the continuous body of the first liquid symmetrically into two sub-bodies of equal volume; and repeatedly dividing each sub-body formed by a preceding dividing step symmetrically into two further sub-bodies of equal volume.
This approach allows multiple sub-bodies of equal volume to be formed accurately and reliably.
In an embodiment, an area of contact between each sub-body and the substrate comprises a sub-body footprint with a sub-body footprint outline; and at least a subset of the sub-body footprint outlines tessellate with respect to each other.
In contrast to prior art methods based on ink jet printing of droplets, embodiments of the present disclosure allow sub-bodies that tessellate with each other to be produced in an efficient manner, thereby achieving high space filling.
In an embodiment, the second liquid is denser than the first liquid.
The method is surprisingly effective using a second liquid that is denser than the first liquid, despite the forces of buoyancy which might be expected to lift the first liquid away from contact with the substrate. Allowing use of a denser second liquid advantageously widens the range of compositions that can be used for the second liquid. Furthermore, the maximum depth of first liquid that can be retained stably in each sub-body without the first liquid spreading laterally over the substrate is increased.
In an embodiment, a material to be investigated is provided in the continuous body of the first liquid, and the division into sub-bodies generates a plurality of isolated samples that each contain a portion of the material to be investigated. In an embodiment, the material to be investigated comprises adherent living cells and at least a portion of the cells are allowed to adhere to the substrate before the continuous body of the first liquid is divided into the sub-bodies. A test substance (e.g. drug) is added to the continuous body of the first liquid after at least a portion of the adherent living cells have adhered to the substrate. The division into the sub-bodies is performed after the test substance has been added to the continuous body of the first liquid.
Thus, a methodology is provided which allows adhered living cells to be treated en masse after they have been allowed to adhere to a substrate and be divided into plural isolated samples later on. This is not possible using prior art approaches and saves considerable time and system complexity, particularly where it is desired to create large numbers of isolated samples and minimum disruption to the cells. It also ensures that cells in each sample have been exposed to very similar conditions, which is difficult to ensure when test substances (e.g. drugs) are added to individual wells or droplets manually, which may impose significant delays between treatment, and physical environments due to inkjet printing or drop-seq method, of different samples. The cells can be placed on the surface without the stresses that would be imposed by passing them through a printing nozzle of an inkjet style printing system. Allowing the cells to adhere before they are cut up provides a better representation of more classical well plate starting conditions for drug screening than alternative approaches in which cells are brought into miniature volumes before they adhere (e.g. via droplet printing). The inventors have furthermore found that cell survival is higher in the sub-bodies formed according to embodiments of the present disclosure in comparison to when the cells were added or present in droplets of the same volume prior to adhesion of the cells.
According to an alternative aspect, there is provided an apparatus for manufacturing a microfluidic arrangement, comprising: an injection system configured to provide a continuous body of a first liquid in direct contact with a substrate by ejecting the first liquid through the distal tip of an injection member while moving the injection member over the substrate to define the shape of the continuous body of the first liquid; a separator system comprising a separator member having a distal tip, the separator system being configured in use to force a second liquid, the second liquid being immiscible with the first liquid, provided in direct contact with the first liquid and covering the first liquid such that the first liquid is in direct contact exclusively with the second liquid and the substrate, through the first liquid and into contact with the substrate in selected regions of the substrate by moving the distal tip of the separator member through the first liquid over the selected regions of the substrate, thereby dividing the continuous body of the first liquid into a plurality of sub-bodies of the first liquid that are separated from each other by the second liquid; and a controller configured to control movement of the injection member over the substrate during the forming of the continuous body of the first liquid and to control movement of the separator member over the substrate during the dividing of the continuous body of the first liquid into the plurality of sub-bodies of the first liquid.
Thus, an apparatus is provided that is capable of performing methods according to the disclosure.
In an embodiment, the injection member and separator member are provided as separate members, allowing optimal properties for the external surfaces of these members to be provided. In an embodiment, at least a portion of the distal tip of the separator member has a surface energy density that is lower in respect of contact with the second liquid than in respect of contact with the first liquid. Preferably, at least a portion of the distal tip of the injection member has a surface energy density that is lower in respect of contact with the first liquid than in respect of contact with the second liquid. The surface properties of the separator member allow the second liquid to follow the movement of the separator member efficiently, thereby displacing the first liquid efficiently. The surface properties of the injection member allow the continuous body of the first liquid to be formed efficiently, even when the continuous body of the first liquid is formed while the second liquid is already present (e.g. by inserting the distal tip through the second liquid to form the continuous body of the first liquid). The surface properties of the injection member also allow the injection member to be used to modify the shape of the first liquid on the substrate after it has been formed (e.g. by spreading the first liquid into new regions on the substrate by dragging the distal tip across the substrate).
Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which corresponding reference symbols indicate corresponding parts, and in which:
The figures are provided for explanatory purposes only and are not depicted to scale in order to allow constituent elements to be visualised clearly. In particular, the width of the receptacle providing the substrate relative to the depth of the first and second fluids will in practice be much larger than depicted in the drawings.
Methods are provided for conveniently and flexibly manufacturing a microfluidic arrangement.
As depicted schematically in
As depicted in
The method allows sub-bodies of the first liquid 1 to be formed flexibly on the substrate 4 without any mechanical or chemical structures being created beforehand to define the geometry of the sub-bodies.
The particular compositions of the first liquid 1, second liquid 2 and substrate 4 are not particularly limited. However, it is desirable that the first liquid 1 and the second liquid 2 can wet the substrate 4 sufficiently for the method to operate efficiently. In an embodiment, the first liquid 1, second liquid 2 and substrate 4 are selected such that an equilibrium contact angle of a droplet of the first liquid 1 on the substrate 4 in air and an equilibrium contact angle of a droplet of the second liquid 2 on the substrate 4 in air would both be less than 90 degrees. In an embodiment, the first liquid 1 comprises an aqueous solution. In this case the substrate 4 could be described as hydrophilic. In an embodiment, the second liquid 2 comprises a fluorocarbon such as FC40 (described in further detail below). In this case the substrate 4 could be described as fluorophilic. In the case where the first liquid 1 is an aqueous solution and the second liquid 2 is a fluorocarbon, the substrate 4 could therefore be described as being both hydrophilic and fluorophilic.
In an embodiment, the forcing of the second liquid 2 through the first liquid 1 is performed by moving a distal tip of a separator member 6 through the first liquid 1 over the selected regions 5 of the substrate 4. The distal tip displaces the first liquid 1 and allows the second liquid 2 to move into the volume previously occupied by the first liquid 1. The second liquid 2 is thereby forced through the first liquid 1. In an embodiment, this process is facilitated by arranging for at least a portion of the distal tip of the separator member 6 to have a surface energy density that is lower in respect of contact with the second liquid 2 than in respect of contact with the first liquid 1. In this way, it is energetically more favourable for the second liquid to flow into the region behind the moving distal tip and thereby displace the first liquid efficiently. Preferably the substrate 4 is also configured so that it is energetically favourable for the second liquid 2 to wet the substrate 4 and thereby remain in contact with the substrate 4 in the selected regions 5 of the substrate 4 and stably hold the first liquid 1 in the separate sub-bodies.
In an embodiment, a sequence of the dividing process is selected to control the relative volumes of the sub-bodies 7 formed. In an embodiment, as depicted in
In an embodiment, an area of contact between each sub-body 7 and the substrate 4 comprises a sub-body footprint with a sub-body footprint outline. At least a subset of the sub-body footprint outlines each comprise at least one straight line portion. This can be achieved for example by forming the sub-bodies using straight line cuts such as those described above with reference to
In an embodiment, the second liquid 2 is denser than the first liquid 1. The inventors have found that despite the buoyancy forces imposed on the first liquid 1 by the denser second liquid 2 above the first liquid 1, the first liquid 1 surprisingly remains stably in contact with the substrate 4 due to surface tension effects between the first liquid 1 and the substrate 4. Allowing use of a denser second liquid 2 is advantageous because it widens the range of compositions that are possible for the second liquid 2. For example, in a case where the first liquid 1 is an aqueous solution, a fluorocarbon such as FC40 can be used, which provides a high enough permeability to allow exchange of vital gases between cells in the sub-bodies 7 and the surrounding atmosphere through the layer of the second liquid 2. FC40 is a transparent fully fluorinated liquid of density 1.8555 g/ml that is widely used in droplet based microfluidics. Using a second liquid 2 that is denser than the first liquid 1 is also advantageous because it increases the maximum depth of first liquid 1 that can be retained stably in each sub-body 7 without the first liquid 1 spreading laterally over the substrate 4. This is because the weight of the first liquid 1 would tend to force the sub-body 7 downwards and therefore outwards and this effect is counteracted by buoyancy.
In the embodiments discussed above the microfluidic arrangement is formed on an upper surface of a substrate 4. In other embodiments, as depicted in
In an embodiment, the continuous body of the first liquid 1 is laterally constrained predominantly by surface tension. For example, the continuous body of the first liquid 1 may be provided only in a selected region on the substrate 4 rather than extending all the way to a lateral wall (e.g. where the substrate 4 is the bottom surface of a receptacle comprising lateral walls, as depicted in
In other embodiments, as depicted schematically in
In an embodiment, the continuous body of the first liquid 1 is divided into a plurality of elongate strips 40 in an initial step of dividing the continuous body of the first liquid 1 into sub-bodies. In an embodiment, the elongate strips 40 are parallel to each other. An example of such an arrangement is depicted in
In an embodiment, more complex shapes can be formed by the dividing of the continuous body of the first liquid 1 into sub-bodies. In one example, as depicted in
In the particular example shown, a T-shaped conduit 36 is provided that connects two source reservoirs 32 and 34 to a sink reservoir 34. Flow is driven in use, e.g. by Laplace pressure, hydrostatic pressure and/or pumping of material into the reservoirs 32, from the source reservoirs 32 to the sink reservoir 34.
In embodiments of the disclosure the continuous body of the first liquid 1 is formed by depositing the first liquid 1 onto the substrate 4 by ejecting the first liquid 1 from a distal tip while moving the distal tip over the substrate 4 to define the shape of the continuous body of the first liquid. This approach may be used for example when forming a continuous body of the first liquid 1 that is laterally constrained predominantly by surface tension (rather than by walls).
As depicted in
As depicted in
In the particular example of
In an embodiment, as depicted schematically in
In an embodiment, the manufactured microfluidic arrangement comprises a plurality of isolated samples that are used for investigating a material of interest. The framework of the method is depicted schematically in
In an embodiment, the material to be investigated comprises biological material. In an embodiment, the biological material comprises adherent living cells. Methods of embodiments of the present disclosure are particularly advantageous in this context because they allow adhered living cells to be treated en masse after they have been allowed to adhere to a substrate 4, and divided into plural isolated samples later on. This is not possible using prior art approaches and saves considerable time and system complexity, particularly where it is desired to create large numbers of isolated samples.
In an embodiment, the above methods are adapted to implement single cell studies. This can be done for example by providing a concentration of living cells in the initial continuous body of the first liquid 1 that is low enough that the mean occupancy of each sub-body created by dividing the continuous body is less than one living cell. In this way, may sub-bodies will be created that contain one and only one cell. This approach is considerably quicker than alternative approaches requiring individual deposition of cells into separate wells after the wells have been created (e.g. in a microwell plate).
The apparatus 30 of
In an embodiment, the apparatus 30 is configured to maintain a small but finite separation between the distal tip of the injection member 18 and the substrate 4 while the injection member 18 is moved over the substrate 4 to form the continuous body of the first liquid 1. This is particularly important where the microfluidic arrangement is to be used for cell-based studies, which would be affected by any scratching or other modification of the surface that might be caused were the injection member 18 to be dragged over the substrate 4 in contact with the substrate 4. Any such modifications could negatively affect optical access and/or cell compatibility. In an embodiment, this is achieved by mounting the injection member 18 slideably in a mounting such that a force from contact with the substrate 4 will cause the injection member 18 to slide within the mounting. Contact between the injection member 18 and the substrate 4 is detected by detecting sliding of the injection member 18 relative to the mounting. When contact is detected, the injection member 18 is pulled back by a small amount (e.g. 20-150 microns) before the injection member 18 is moved over the substrate 4 to form the continuous body of the first liquid 1 (without contacting the substrate 4 during this motion). This approach to controlling separation between the distal tip and the substrate 4 can be implemented cost effectively in comparison to alternatives such as the capacitive/inductive methods used in 3D printers, or optical based sensing techniques. The approach also does not require a conductive surface to be provided.
The apparatus 30 of
The apparatus 30 of
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| Number | Date | Country | |
|---|---|---|---|
| 20190176148 A1 | Jun 2019 | US |
