GB2569328A

GB2569328A - Methods and apparatus for manufacturing a microfluidic arrangement , and a microfluidic arrangement

Info

Publication number: GB2569328A
Application number: GB1720771.3A
Authority: GB
Inventors: Walsh Edmond; Richard Cook Peter; Soitu Cristian
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2019-06-19
Also published as: GB201720771D0; WO2019116033A1

Abstract

A method of manufacturing a microfluidic arrangement comprises modifying a selected region of a substrate surface 14 by moving a probe over the selected region while maintaining contact between the probe and the substrate surface to create a modified region 60. Modification causes an equilibrium contact angle of a first liquid 11, when disposed at the selected region, to be to be higher after modification than before. The modified region forms one or more unmodified isolated regions 64 on the substrate surface by surrounding each isolated region with a closed path of modification. Ideally, a hydrophilic substrate 10 has selected region modification to create hydrophobic or less hydrophilic regions which surround isolated hydrophilic regions or pockets. The method further comprises forming a microfluidic arrangement comprising a first liquid body 6, 11 in direct contact with each isolated region, and a second liquid 12 in direct contact with, and covering, each first liquid body and the modified region, wherein the second liquid is immiscible with the first liquid, and each first liquid body is held in shape predominantly by surface tension and substantially fills each isolated region. An apparatus for manufacturing the arrangement comprises a substrate, scanning device, probe, dispensing system and controller.

Description

(54) Title of the Invention: Methods and apparatus for manufacturing a microfluidic arrangement, and a microfluidic arrangement Abstract Title: Methods and apparatus for manufacturing a microfluidic arrangement (57) A method of manufacturing a microfluidic arrangement comprises modifying a selected region of a substrate surface 14 by moving a probe over the selected region while maintaining contact between the probe and the substrate surface to create a modified region 60. Modification causes an equilibrium contact angle of a first liquid 11, when disposed at the selected region, to be to be higher after modification than before. The modified region forms one or more unmodified isolated regions 64 on the substrate surface by surrounding each isolated region with a closed path of modification. Ideally, a hydrophilic substrate 10 has selected region modification to create hydrophobic or less hydrophilic regions which surround isolated hydrophilic regions or pockets. The method further comprises forming a microfluidic arrangement comprising a first liquid body 6, 11 in direct contact with each isolated region, and a second liquid 12 in direct contact with, and covering, each first liquid body and the modified region, wherein the second liquid is immiscible with the first liquid, and each first liquid body is held in shape predominantly by surface tension and substantially fills each isolated region. An apparatus for manufacturing the arrangement comprises a substrate, scanning device, probe, dispensing system and controller.

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70

METHODS AND APPARATUS FOR MANUFACTURING A MICROFLUIDIC ARRANGEMENT,

AND A MICROFLUIDIC ARRANGEMENT

The invention relates to manufacturing a microfluidic arrangement.

Manipulation of small volumes of liquids is central to many scientific disciplines, including microbiology, cell biology, biochemistry, and materials science. Microfluidic devices in which liquid flows through channels in polydimethylsiloxane (PDMS) are available but fewer such devices than expected have been incorporated into scientific workflows despite demonstrated advantages of the technology. Various reasons are given. Prototyping PDMS-based devices takes at least a few days and is expensive; it also typically requires specialized equipment, a clean room, and advanced training. Once made, devices are usually dedicated to one application, and access to most points in them is limited. Moreover, untreated PDMS has poor biological and chemical compatibility because it leaches toxins and reacts with organic solvents. Air bubbles in conventional devices also present numerous operational challenges: they unbalance flows, damage incorporated cells, and trigger molecular aggregation at airfluid interfaces.

It is an object of the invention to improve manufacture of microfluidic arrangements.

According to an aspect of the invention, there is provided a method of manufacturing a microfluidic arrangement, comprising: modifying a selected region of a substrate surface by moving a probe over the selected region of the substrate surface while maintaining contact between the probe and the substrate surface, wherein the modification is such that, if a first liquid were provided over the modified selected region, an equilibrium contact angle of the first liquid with respectto contact with the modified selected region would be higher than without the modification, wherein: the modified selected region forms one or more isolated regions on the substrate surface by surrounding each isolated region, the modified selected region forming a closed path around each isolated region; and the method further comprises forming a microfluidic arrangement comprising a body of the first liquid in direct contact with each isolated region, and a second liquid in direct contact with, and covering, each body of first liquid and the modified selected region of the substrate surface, wherein the second liquid is immiscible with the first liquid, and each body of first liquid is held in shape predominantly by surface tension and substantially fills each isolated region.

Thus, a method is provided in which a microfluidic arrangement without solid walls is formed. Surface tension is used instead of solid walls to hold a first liquid in a desired microfluidic pattern. Because the substrate does not have to be processed in the same way as PDMS to provide channels or wells, the substrate can be made from materials of proven biocompatibility, such as the polystyrene/glass dishes that biologists commonly use. Furthermore, the risk of contamination of the first liquid is reduced or eliminated by the second liquid. The risk of seal failure or leakage is reduced. If bubbles of gas arise they will be forced up and out of the microfluidic pattern by buoyancy forces, thereby eliminating also the negative effects of bubbles that are regularly encountered in PDMS-based devices and existing high density well plates where bubbles are held in place due to interfacial forces with the physical walls.

Defining the shapes of the body or bodies of first liquid by moving a probe over the substrate surface while maintaining contact between the probe and a selected region provides an efficient and flexible way to define the geometry of the microfluidic arrangement. The geometry of the microfluidic arrangement can be defined before any liquid is added to the substrate surface but without needing solid walls to define channels or wells. Furthermore, no complex chemical or plasma-based treatment of the substrate surface is needed to provide the required modification of surface energy properties. A simple and easily controlled mechanical action of moving an element over the surface is adequate. The methodology can thus be implemented using a cost effective, reliable and/or compact apparatus.

According to an alternative aspect, there is provided an apparatus for manufacturing a microfluidic arrangement, comprising: a substrate; a scanning device configured to scan a probe over a substrate surface of the substrate; a dispensing system configured to output liquid from a port onto the substrate surface; and a controller configured to control the scanning device such that the probe is moved over a selected region of the substrate surface while maintaining contact between the probe and the substrate surface, wherein: the probe, the substrate surface, and the scanning are configured such that a selected region of the substrate surface is modified by the moving of the probe over the selected region, wherein the modification is such that, if a first liquid were provided over the modified selected region, an equilibrium contact angle of the first liquid with respect to contact with the modified selected region would be higher than without the modification; the modified selected region forms one or more isolated regions on the substrate surface by surrounding each isolated region, the modified selected region forming a closed path around each isolated region; the controller is further configured to control the dispensing system to form a microfluidic arrangement comprising a body of the first liquid in direct contact with each isolated region, and a second liquid in direct contact with, and covering, each body of first liquid and the modified selected region of the substrate surface, wherein the second liquid is immiscible with the first liquid, and each body of first liquid is held in shape predominantly by surface tension and substantially fills each isolated region.

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:

Figure 1 schematically depicts an apparatus for manufacturing a microfluidic arrangement;

Figure 2 is a schematic top view showing a scanning path of a probe over a selected region of a substrate surface to form isolated regions;

Figure 3 is a schematic side view of a microfluidic arrangement formed using the surface modification shown in Figure 2;

Figures 4-8 are schematic side views showing steps in a method of manufacturing a microfluidic arrangement;

Figures 9-12 are schematic side views showing steps in an alternative method of manufacturing a microfluidic arrangement;

Figure 13 is a schematic side view depicting how second liquid can be made to move down a connection path comprising all or part of a side wall of the substrate 10 and into contact with a selected region on a bottom interior surface of the substrate;

Figure 14 is a schematic side view depicting how second liquid can be made to move down a probe and into contact with a selected region on a bottom interior surface of the substrate;

Figure 15 is a schematic side view of a microfluidic arrangement formed using the approach depicted in Figure 13 or 14;

Figures 16-18 are schematic side views showing formation of isolated bodies of first liquid by shaking a substrate;

Figures 19-23 are schematic side views showing an alternative method of manufacturing a microfluidic arrangement; and

Figure 24 is atop view of an alternative body of first liquid comprising conduits and reservoirs.

The figures are provided for explanatory purposes only and are not depicted to scale in order to allow different elements to be visualised clearly. In particular, the bodies of the first liquid are depicted much larger relative to other elements of apparatus than would typically be the case in practice.

Figure 1 depicts an apparatus 2 for manufacturing a microfluidic arrangement 4. An example of a microfluidic arrangement 4 is shown in Figure 3 and described in further detail below. The apparatus 2 comprises a scanning device 20. The scanning device 20 is configured to scan a probe 50 over a substrate surface 14 of a substrate 10. The scanning device 20 can be implemented in various ways known in the art of mechanical scanning. The scanning device 20 may for example comprise an electrical motor, one or more sensors for sensing a position of the probe 50, and a gantry or other mounting arrangement for moveably mounting the probe 50 so that it can be driven between different positions by the motor.

The apparatus 2 further comprises a dispensing system 24 configured to output liquid from a port 22 onto the substrate surface 14. The dispensing system 24 typically comprises a pumping system, one or more reservoirs for holding material or components of material to be dispensed via the port 22, and a suitable system of conduits and/or valves for conveying the material from the one or more reservoirs to the port 22.

In an embodiment, as depicted schematically in Figure 1, the scanning device 20 is configured to selectively lower and raise the probe 50 and the port 22 as needed, so that the probe 50 can be positioned in contact with the substrate surface 14 during scanning of the probe 50 over the substrate surface 14 to form the selected region 60 while the port 22 is held in a raised position (as depicted in Figure 1), and the port 22 can be lowered to be positioned appropriately (e.g. a small distance above the substrate surface 14) for outputting liquid in order to form the microfluidic arrangement 4 while the probe 50 is held in a raised position. In an embodiment, the probe 50 and the port 22 are movably mounted in the same head, so as to be scanned together over the substrate 10 as aunit. Alternatively, the probe 50 and the port 22 maybe mounted in such a way that the probe 50 can be scanned over the substrate 10 while the port 22 is held stationary. This may be appropriate for example where the port 22 does not need to be moved over the substrate 10 at all or not in the same way as the port 22.

The port 22 may take various forms, but will typically be tubular and comprise an internal lumen for conveying material to or from a distal end where material can enter or leave the port 22. The port 22 may be configured such that a selected portion of the outer surface of the port 22 proximate to the distal end of the port 22 has a surface energy density in respect of contact with the first liquid 11 (i.e. a surface energy per unit area of an interface between the surface and the first liquid) that is higher than in respect of contact with the second liquid 12. It is therefore energetically more favourable for the second liquid 12 to wetthe selected portion ofthe port 22 than for the first liquid 11 to wetthe selected portion of the port 22. In the case where the first liquid 11 contains a high proportion of water, the selected portion may be configured to be hydrophobic for example. Configuring the port 22 in this way prevents unwanted wetting of the port 22 by liquid leaving the distal end, thereby promoting for example reliable formation of a globule that protrudes towards substrate 10. In an embodiment, the selected portion forms a closed ring around the port 22. The selected portion may comprise an end surface ofthe port 22, a side surface of the port 22, or both. The selected portion may comprise a region that is directly adjacent to the distal end of the port 22. The selected portion may be partially or completely implemented by treating a surface of the port 22, for example by coating, or by providing a port 22 comprising an inner element and a sleeve over the inner element, wherein an outer surface of the sleeve provides the selected portion. In an embodiment, the port 22 comprises a stainless steel inner element and a PTFE sleeve or coating over the inner element.

The apparatus 2 further comprises a controller 28 for controlling the scanning device 24 such that the probe 50 is moved over a selected region 60 (see Figures 2 and 3) of the substrate surface 14 while maintaining contact between the probe 50 and the substrate surface 14. The controller 28 may be implemented using standard data processing hardware (e.g. CPU, bus, memory, etc.), firmware and/or software. The probe 50, the substrate surface 14, and the scanning are configured such that a surface energy density of the substrate surface 14 with respect to contact between the substrate surface 14 and a first liquid is modified in the selected region 60 of the substrate surface 14. In an embodiment, the modification is such that if a first liquid were provided over the modified selected region, an equilibrium contact angle of the first liquid with respect to contact with the modified selected region would be higher than without the modification.

Surface energy density represents a surface energy per unit area associated with the interface defined by the surface. The first liquid does not have to be present when the selected region 60 is modified. The reference to the first liquid is made to describe the nature of the modification. The surface energy density of the substrate surface 14 with respect to contact between the substrate surface 14 and the first liquid thus represents the surface energy per unit area of the inte rface between the substrate 10 and the first liquid, if the first liquid were present, at the substrate surface 14. In a case where the first liquid is aqueous for example, a hydrophilic substrate surface 14 would have a relatively low surface energy density with respectto contact with the first liquid. A hydrophobic substrate surface 14, in contrast, would have a higher surface energy density with respect to contact with the first liquid for an aqueous first liquid.

Figures 2 and 3 depict an example modified selected region 60. Figure 2 is atop view showing formation of the modified selected region 60 partway through the process, such that the modified selected region 60 is not fully complete. The arrowed broken lines indicate a direction and geometry of a scanning path of the probe 50. The scanning path started at position A and has so far reached position B. Inthis particular example, the probe 50 is made to contact the substrate surface 14 only along straight line segments and is removed from the substrate surface 14 during curved segments which join one scanning line to a neighbouring scanning line. The contact between the probe 50 and the substrate surface 14 is thus maintained at least during periods when the probe 50 is moving over the selected region 60 to be modified, but this need not be achieved without temporarily breaking the contact in order for the probe 50 to move from one part of the selected region 60 to another part of the selected region 60. In other embodiments, the probe 50 may be held in contact with the substrate surface 14 continually during the whole scanning process.

The modified selected region 60 shown in Figures 2 and 3 is an example of a modified selected region 60 that forms at least one isolated region 64 on the substrate surface 14. At the stage of the process shown in Figure 2, six isolated regions 64 have been formed. Each isolated region 64 is isolated in the sense of being surrounded by a portion of the modified selected region 60 forming a closed path. In the example shown, the closed path around each isolated region 64 is square, but any other shape may be used. Closed paths that tessellate with each other are advantageous because they allow isolated regions 64 to be formed in a closely packed manner and/or with efficiently realisable scanning paths of the probe 50. In the particular example shown in Figure 2, for example, isolated regions 64 are formed by a scanning path (depicted by broken lines) comprising a first raster (serpentine) scan parallel to a first direction (horizontal in the orientation shown in Figure 2) followed by a second raster scan parallel to a second direction (vertical in the orientation shown in Figure 2), such that the first and second raster scans intersect each other and thereby form the isolated regions 64. The first and second directions may be perpendicular to each other (to form square or rectangular isolated regions) or may be arranged at an oblique angle relative to each other.

The modification of the substrate surface 14 by the movement of the probe 50 in contact with the substrate surface 14 increases the surface energy density (increases an equilibrium contact angle) with respect to contact with the first liquid. Thus, in the case where the substrate surface 14 was hydrophilic to begin with and the first liquid is aqueous, the modification may cause the substrate surface 14 to become less hydrophilic or even hydrophobic in the modified selected region 60.

As exemplified by Figure 3, the controller 28 is further configured to control the dispensing system 24 to provide a body 6 of first liquid 11 in direct contact with each isolated region 64. The controller 28 further causes the dispensing system 24 to provide a second liquid 12 in direct contact with, and covering, each body 6 of first liquid 11. The bodies 6 of first liquid 11 may be provided before or after the second liquid 12. The second liquid 12 is also in contact with the modified selected region 60. The process is controlled so that a microfluidic arrangement 4 is thereby formed in which each body 6 of first liquid 11 is held in shape predominantly by surface tension and substantially fills the isolated region 64. Thus, each isolated region 64 is in contact exclusively with first liquid 11 and not any second liquid 12. In an embodiment, the portion of the substrate surface 14 underneath and immediately adjacent to each body 6 of first liquid 11 is planar and the shape of the body 6, apart from the lower planar extremity of the body 6, where the body 6 is in contact with the substrate surface 14, is determined predominantly by surface tension (rather than by solid walls of a well, as would the case in a traditional microwell plate). In an embodiment, the substrate surface 14 underneath a plurality of the bodies 6 is continuously planar underneath those bodies 6. In an embodiment, the substrate surface 14 is continuously planar under all bodies 6 of first liquid 11 present on the substrate 10. Typically, the substrate surface 14 will be substantially planar and unpattemed (neither mechanically nor chemically), at least in regions where the bodies 6 are provided, prior to brushing by the probe 50.

The second liquid 12 isolates each body 6 from each other body 6. Thus, the second liquid 12 is present in a region between each body 6 and each other body 6. In an embodiment, the second liquid 12 is provided as a single continuous body of second liquid, such that every point in the second liquid 12 is connected to every other point in the second liquid 12 by a continuous path exclusively through second liquid. Typically, each of the bodies 6 of the first liquid 11 will be in contact exclusively with the substrate surface 14 and the second liquid 12. The first liquid 11 in each body 6 is thus in contact with the substrate 10 on one side and with the second liquid 12 on all of the rest of an outer interface of the body 6. The second liquid 12 isolates the first liquid 11 from the surrounding environment (e.g. air). The second liquid 12 may therefore reduce or prevent evaporation of the first liquid 11. The second liquid 12 reduces or prevents contamination of the first liquid 11. The second liquid 12 is substantially immiscible with the first liquid 11. In an embodiment, the second liquid 12 is denser than the first liquid 11.

In an embodiment, the modification of the surface energy density is such that the surface energy density in the selected region 60 of the substrate surface 14 is higher with respect to contact with the second liquid 12 than in respect of contact with the first liquid 11 before the modification and is lower with respect to contact with the second liquid 12 than in respect of contact with the first liquid 11 after the modification. The unmodified surface energy density of the substrate surface 14 in regions outside of the selected region 60 is lower in respect of contact with the first liquid 11 than in respect of contact with the second liquid 12. Thus, in an embodiment the modification of the selected region is such that an equilibrium contact angle of the first liquid with respect to contact with the modified selected region is higher than an equilibrium contact angle of the second liquid with respect to the modified selected region, and an equilibrium contact angle of the first liquid with respect to contact with the selected region before the modification is lower than an equilibrium contact angle of the second liquid with respect to contact with the selected region before the modification.

In an embodiment, the substrate surface 14 prior to modification comprises a surface treatedtobe hydrophilic and the modification causes the selected region 60 of the substrate surface 14 to become less hydrophilic or hydrophobic.

In an embodiment, the moving of the of the probe 50 over the selected region 60 of the substrate surface 14 modifies the surface energy density at least partly by depositing material on the substrate surface 14. In an embodiment, the material is deposited due to friction causing detachment of the material from the probe 50. Thus, the probe 50 may gradually wear away during the scanning of the probe 50, leaving a trail of deposited material along a scanning path that is effective to modify the surface energy density in the desired manner. In an embodiment, the probe 50 is formed from a single material (e.g. PTFE) or coated with a coating formed from a single material, and the detachment of material comprises detachment of the single material. In an embodiment, the probe 50 is formed from, or coated with, a hydrophobic material such as PFTE, at least part of which is progressively deposited onto the substrate surface 14 during the movement of the probe 50. Alternatively, the material is deposited merely by contact between the surfaces, with further material being conveyed to a tip of the probe as needed, for example by wicking in the manner of a felt-tipped pen. In an embodiment the material is deposited in liquid form and evaporates to leave a residue.

Alternatively or additionally, the movement of the probe 50 over the selected region 60 of the substrate surface 14 modifies the surface at least partly by removing material from the substrate surface 14 by friction with the surface or by cutting into the substrate surface 14. The probe 50 may thus cause the modification in surface energy density by disrupting or removing a surface state of, or a thin coating on, the substrate surface 14, which may have been modified or prepared previously to have particular surface energy properties (e .g. to be more hydrophilic than a base material forming the rest of the substrate 10). The movement of the probe 50 may even be such as to cut into the substrate surface 14, for example to cause shallow grooves in the substrate surface 14.

Thus, a method is provided in which a substrate surface 14 is modified in a selected region 60 of the substrate surface 14. The modification is achieved by moving a probe 50 over the selected region 60 of the substrate surface 14 while maintaining contact between the probe and the substrate surface 14. The modified selected region 60 forms at least one isolated region 64 on the substrate surface 14. The isolated region 64 is surrounded by a portion of the modified selected region 60 forming a closed path. The modification is such that, if a first liquid were provided over the modified selected region 60, an equilibrium contact angle of the first liquid 11 with respect to contact with the modified selected region 60 would be higher than without the modification. In an embodiment, the modification of the surface energy density comprises increasing the surface energy density. A body 6 of first liquid 11 is provided in direct contact with the isolated region 64. A second liquid 12 is provided in direct contact with, and covering, the body 6 of first liquid 11 and the modified selected region 60 of the substrate surface 14. The body 6 of first liquid 1 is held in shape predominantly by surface tension and substantially fills the isolated region 64.

Figures 4-8 exemplify one approach for performing the method.

In a first step, as depicted in Figure 4, a substrate 10 is provided that has a substantially uniform substrate surface 14. The substrate surface 14 may comprise for example a substantially planar surface that has been treated to be hydrophilic, such as by a plasma treatment. Such a substrate surface 14 may be provided for example by a vessel designed to receive cell cultures, such as a petri dish. In an embodiment, the substrate surface 14 is provided by an interior surface of a vessel, optionally a cell culture dish, that has undergone surface treatment to make the surface hydrophilic, for example by plasma treatment (e .g. a dish made from a hydrophobic material such as polystyrene and treated to become hydrophilic using corona discharge under atmospheric conditions or gas-plasma under vacuum).

In a second step, as depicted in Figure 5, a modified selected region 60 is formed by moving a probe 50 over the substrate surface 14 to form isolated regions, as discussed above.

In a third step, as depicted in Figure 6, a layer of the first liquid 11 is provided that completely covers the modified selected region 60 and the (unmodified ) isolated region or isolated regions formed by the modified selected region 60 (and any other unmodified regions).

In a fourth step, as depicted in Figure 7, the first liquid is removed (e.g. by sucking the liquid away or by controlled pouring) until bodies 6 of the first liquid 11 are formed in regions corresponding to the isolated regions, separated from each other by portions of the modified selected region 60 formed by the movement of the probe 50.

In a fifth step, as depicted in Figure 8, the second liquid 12 is added to complete the microfluidic arrangement 4. The second liquid 12 is in direct contact with, and covering, each body 6 of first liquid 11 that is in contact with one of the isolated regions. The second liquid 12 covers the modified selected region 60 of the substrate surface 14.

Material can be added to or removed from each body 6 individually by inserting a suitably aligned port through the second liquid 12 and pumping material out of the port into the body 6 or by sucking material into the port from the body 6. The second liquid 12 advantageously acts as a self-sealing arrangement in this process, reducing the risk of contamination of each body 6 from the environment or from material from, or intended for, other bodies 6. If it is desired to add the same material to all bodies 6, this can be achieved efficiently by removing all of the second liquid 12, forming a layer of the material to be added over the top of the bodies 6 (such that the bodies 6 are no longer isolated from each other), to arrive at an arrangement resembling that of Figure 6 described above. The material to be added and the first liquid may both be aqueous for example. The fourth and fifth steps described above with reference to Figures 7 and 8 canthen be repeated to arrive again at a microfluidic arrangement such as that depicted in Figure 8.

Figures 9-12 exemplify an alternative approach for performing the method.

In a first step, as depicted in Figure 9, a substrate 10 is provided that has a substantially uniform substrate surface 14. The substrate surface 14 may comprise for example a substantially planar surface that has been treated to be hydrophilic, such as by a plasma treatment. Such a substrate surface 14 may be provided for example by a vessel designed to receive cell cultures, such as a petri dish.

In a second step, as depicted in Figure 10, a modified selected region 60 is formed by moving a probe 50 over the substrate surface 14 to form isolated regions, as discussed above.

In a third step, as depicted in Figure 11, a layer of the first liquid 11 is provided that completely covers the modified selected region and the (unmodified) isolated region or isolated regions formed by the modified selected region (and any other unmodified regions).

In a fourth step, as depicted in Figure 12, the second liquid 12 is added to complete the microfluidic arrangement 4. The second liquid 12 is added in such a way that the second liquid 12 displaces the first liquid 11 where the first liquid 11 was in contact with the modified selected region 60, such that the second liquid 12 is in direct contact with the modified selected region 60, thereby isolating the bodies 6 of the first liquid 11 that are in direct contact with one of the isolated regions, such that each body 6 is in contact exclusively with the second liquid 12 and the substrate 10. The second liquid 12 may be added by injecting a flow of the second liquid 12 onto the modified selected region 60 of the substrate surface 14. The second liquid 12 then moves along the modified selected region 60 of the substrate surface 14 until all of the modified selected region 60 is covered by the second liquid 12 and the microfluidic arrangement is formed 4. In this case, the first liquid may be pushed to one side of the microfluidic arrangement 4 and may even rise up above the second liquid 12, as depicted schematically in Figure 12 on the right hand side, and/or spread over the top of the second liquid 12.

In an alternative embodiment, a layer of the second liquid 12 (denser than the first liquid 11) is provided on top of the layer of first liquid 11 in the arrangement of Figure 11 to provide the arrangement shown in Figure 13. The side walls 80 of the substrate 10 and a portion of the surface 14 of the substrate 10 are configured to provide a connection path between the second liquid 12 on top of the first liquid 11 and the modified selected region 60. The connection path is such that the second liquid 12 wets the connection path in preference to the first liquid 11. Thus, the connection path is such that a surface energy density in the connection path is higher in respect of contact with the first liquid 11 than in respect of contact with the second liquid 12. This arrangement promotes movement of the second liquid 12, as depicted schematically in Figure 13, down the side walls 80 (along the connection path) and into contact with the modified selected region 60 of the substrate surface 14. The second liquid 12 thereafter moves along the modified selected region 60 until all of the modified selected region 60 is covered by the second liquid 12, which separates the first liquid 11 into isolated bodies 6, as depicted in Figure 15.

In a further alternative embodiment, a layer of the second liquid 12 (denser than the first liquid 11) is again provided on top of the layer of first liquid 11 in the arrangement of Figure 11, but instead of using a connection path along the sides walls 80, a probe 82 is used to provide an energetically favourable path for second liquid 12 to pass through the first liquid 11 and contact the selected region 60, as depicted in Figure 14. At least a portion of the outer surface of the probe 82 is such that the second liquid 12 wets the probe 82 in preference to the first liquid 11. Thus, the probe 82 is such that a surface energy density on the outer surface of the probe 82 is higher in respect of contact with the first liquid 11 than in respect of contact with the second liquid 12. This arrangement promotes movement of the second liquid 12 as depicted schematically in Figure 14 down the probe 82 and into contact with the modified selected region 60 of the substrate surface 14. The second liquid 12 thereafter moves along the modified selected region 60 until all of the modified selected region 60 is covered by the second liquid 12, which separates the first liquid 11 into isolated bodies 6, as depicted in Figure 15. In embodiments in which the first liquid 11 is aqueous, the probe 82 may have a hydrophobic outer surface. The probe 82 may be formed from PTFE or comprise a PFTE coating or sleeve for example.

Figure 16-18 depict a still further variation. In this embodiment, a substrate surface Mis modified by moving a probe 50 over a selected region 60 to form isolated regions, as discussed above. A pool of first liquid 11 is then provided on the substrate surface 14 as shown in Figure 16. The pool of first liquid 11 may be provided outside of or overlapping with a region of the substrate surface 14 patterned by the probe 50. It has been found that shaking the substrate 10, as depicted schematically in Figure 17, causes the first liquid 11 to move into the isolated regions defined by the modified selected region 60 and thereby forms aplurality of isolated bodies 6 of first liquid 11. Secondliquid 12 canthen be added over the top to form a microfluidic arrangement 4 as depicted in Figure 18.

Figures 19-23 depict a still further variation.

In a first step, as depicted in Figure 19, a substrate 10 is provided that has a substantially uniform substrate surface 14. The substrate 10 may be configured as described above with reference to Figure 4.

In a second step, as depicted in Figure 20, a modified selected region 60 is formed by moving a probe 50 over the substrate surface 14 to form isolated regions, as discussed above.

In a third step, as depicted in Figure 21, first liquid 11 and one or more first components dissolved or suspended in the first liquid 11 are added to each isolated region to form bodies 6A of first liquid 11.

In a fourth step, the first liquid 11 is dried to leave a residue in each isolated region comprising the one or more components.

In a fifth step, a layer of the second liquid 12 is provided to cover the modified selected region 60 and each isolated region, thereby provided the arrangement depicted in Figure 22.

In a sixth step, as depicted in Figure 23, first liquid 11 and one or more second components dissolved or suspended in the first liquid 11 are added to each isolated region, through the second liquid 12. A microfluidic arrangement 4 is thereby formed that comprises bodies 6B of the first liquid 11 comprising the first liquid 11 and the first and second components dissolved or suspended in, or at least in contact with, the first liquid 11.

Thus, a methodology is provided that allows a modified selected region to be formed that provides the desired surface energy (contact angle) characteristics in a flexible and convenient manner, via a residue formed by drying bodies 6A to leave the first components. Bodies 6B containing first liquid 11 and second components suitable for performing a desired experiment, including for example biological cells and experimental reagents, can be formed underneath a protective second liquid 12 when it is desired to commence the experiment. This can be achieved by injecting the first liquid and second components through the second liquid 12 using a needle for example. Alternatively, the second components or the second components and a portion of the first liquid 11 could be injected via a needle, and first liquid, or additional first liquid 11, could be provided by exposure to an atmosphere in which the first liquid is present in vapour form at a high concentration (e.g. a humid atmosphere when the first liquid is aqueous). The presence of the second liquid 12 protects the bodies 6B from contamination and/or prevents or reduces evaporation of the first liquid 11. This approach helps to ensure that experimental conditions are constant between different bodies 6B (e.g. same amount of first liquid 11 is present). In an embodiment, the first components comprise one or more of the following: siRNA, gRNA, DNAor RNA bar-codes, drugs, sugars, salts, coated beads, magnetic beads, antibodies, growth factors. In an embodiment, the second components comprise one or more of the following: biological cells, reagents, growth factors, cytokines, DNA-or RNA-based bar codes, antibodies, drugs serum-free media, cell medium and serum, phosphate-buffer saline, water.

In the above examples, the microfluidic arrangement 4 comprises multiple localized bodies 6 of the first liquid 11 arranged in a regular grid. Such an arrangement may be used for example in place of a traditional microwell plate, with each body of first liquid 11 being able to act as an individual well. Liquid can be added to or removed from each of the bodies 6 without the footprint of each body 6 changing. Changes in the volume of each body 6 can be accommodated by a change in the contact angle between the first liquid 11 and the substrate surface 14 between a static receding contact angle and a static advancing contact angle. The provision of an array of bodies 6 acting as wells but separated from each other by liquid rather than solid walls is convenient because it can be formed flexibly (the number and size of the bodies 6 can be varied according to need), the bodies 6 can be made smaller and/or fitted closer together than is typically possible where solid walls are needed to separate wells, and problems associated with bubbles are avoided (bubbles cannot be trapped within the bodies 6 in the same way as they can in solid wells).

The above methodology can be used, however, to form a variety other types of microfluidic arrangements 4. An example arrangement is shown in Figure 24, for example, where a microfluidic arrangement is shown comprising a body 6 of the first liquid 11 shaped so as to define two input reservoirs 70, elongate conduits 71 in an inverted Y-shape, and a sink reservoir 72. This is an example of a microfluidic circuit, in which liquids added to the microfluidic arrangement 4 can be made to flow from the one or more input points in the circuit (in this case, the two input reservoirs 70) to one or more output points or sinks (in this case, the sink reservoir 71). Such microfluidic arrangements 4 can be made more flexibly than the closest equivalent using solid walls, do not need to use materials such as PDMS, which can leach toxins or react with organic solvents, and are less prone to problems caused by trapped air bubbles.

The particular compositions of the first liquid 11, second liquid 12 and substrate 10 are not particularly limited, as long as the above-de scribed surface energy (contact angle) characteristics are obtained. In some embodiments, the first liquid 11 is aqueous (i.e. predominantly comprises water). In some embodiments, the second liquid 12 comprises a fluorocarbon such as FC40. FC40 provides a high enough permeability to allow exchange of vital gases between cells in the bodies 6 and the surrounding atmosphere through the layer of the second liquid 12. FC40 is a transparent fully fluorinated liquid of density 1.8555 g/ml that is widely used in droplet based microfluidics. In an embodiment, the second liquid 12 is denser than the first liquid 11 (as would be the case where the second liquid 12 predominantly comprises FC40 and the first liquid 11 is aqueous). The inventors have found that despite the buoyancy forces imposed on the first liquid 11 by the denser second liquid 12 above the first liquid 11, the first liquid 11 surprisingly remains stably in contact with the substrate 10 due to surface tension effects between the first liquid 11 and the substrate 10. Using a second liquid 12 that is denser than the first liquid 11 is advantageous because it increases the maximum depth of first liquid 11 that can be retained stably in each body 6 without the first liquid 11 spreading laterally over the substrate 10. This is because the buoyancy of first liquid 11 in the body 6 tends to drive the first liquid 11 upwards instead of laterally over the substrate 10.

Claims

1. A method of manufacturing a microfluidic arrangement, comprising:

modifying a selected region of a substrate surface by moving a probe over the selected region of the substrate surface while maintaining contact between the probe and the substrate surface, wherein the modification is such that, if a first liquid were provided over the modified selected region, an equilibrium contact angle of the first liquid with respect to contact with the modified selected region would be higher than without the modification, wherein:

the modified selected region forms one or more isolated regions on the substrate surface by surrounding each isolated region, the modified selected region forming a closed path around each isolated region; and the method further comprises forming a microfluidic arrangement comprising a body of the first liquid in direct contact with each isolated region, and a second liquid in direct contact with, and covering, each body of first liquid and the modified selected region ofthe substrate surface, wherein the second liquid is immiscible with the first liquid, and each body of first liquid is held in shape predominantly by surface tension and substantially fills each isolated region.

2. The method of claim 1, wherein the modified selected region forms a plurality of the isolated regions on the substrate surface.

3. The method of claim 2, wherein the body of first liquid in each isolated region is separated from the body of first liquid in each other isolated region by the second liquid.

4. The method of any preceding claim, wherein the forming of the microfluidic arrangement comprises the following steps in order:

providing a layer of the first liquid covering the modified selected region and each isolated region;

removing first liquid until the body or bodies of first liquid are formed; and providing the second liquid in direct contact with, and covering, each body of first liquid that is in contact with one of the isolated regions, thereby forming the microfluidic arrangement.

5. The method of any of claims 1-3, wherein the forming of the microfluidic arrangement comprises the following steps in order:

adding the second liquid and arranging for the second liquid to displace the first liquid where the first liquid was in contact with the modified selected region, such that the second liquid is in direct contact with the modified selected region, thereby isolating each body of first liquid that is in direct contact with one of the isolated regions, such that each body is in contact exclusively with the second liquid and the substrate, thereby forming the microfluidic arrangement.

6. The method of any of claims 1-3, wherein the forming of the microfluidic arrangement comprises the following steps in order:

adding to each isolated region first liquid and one or more first components dissolved or suspended in the first liquid;

drying the first liquid to leave a residue in each isolated region comprising the one or more components;

providing a layer of the second liquid covering the modified selected region and each isolated region; and adding to each isolated region, through the second liquid, first liquid and one or more second components dissolved or suspended in the first liquid, thereby forming the microfluidic arrangement.

7. The method of claim 6, wherein the first components comprise one or more of the following: siRNA, gRNA, DNA or RNAbar-codes, drugs, sugars, salts, coated beads, magnetic beads, antibodies, growth factors.

8. The method of claim 6 or 7, wherein the second components comprise one or more of the following: biological cells, reagents, growth factors, cytokines, DNA-or RNA-based bar codes, antibodies, drugs serum-free media, cell medium and serum, phosphate-buffer saline, water.

9. The method of any preceding claim, wherein the modification of the selected region is such that: an equilibrium contact angle of the first liquid with respect to contact with the modified selected region is higher than an equilibrium contact angle of the second liquid with respect to the modified selected region; and an equilibrium contact angle of the first liquid with respect to contact with the selected region before the modification is lower than an equilibrium contact angle of the second liquid with respect to contact with the selected region before the modification.

10. The method of claim 9, wherein an equilibrium contact angle of the first liquid with respect to contact with regions outside of the modified selected region is lower than an equilibrium contact angle of the second liquid with respect to contact with the regions outside of the modified selected region.

11. The method of any preceding claim, wherein the substrate surface prior to modification comprises a surface treated to be hydrophilic.

12. The method of claim 11, wherein the substrate surface prior to modification comprises a surface uniformly treated to be hydrophilic over a region which contains all of the selected region to be modified.

13. The method of any preceding claim, wherein the modification of the selected region is achieved at least partly by depositing material from the probe onto the substrate surface.

14. The method of claim 13, wherein the material is deposited due to friction causing detachment of the material from the probe.

15. The method of claim 14, wherein the probe is formed from a single material, or a coating formed from a single material, and the detachment of material comprises detachment of the single material.

16. The method of any preceding claim, wherein the modification of the selected region is achieved at least partly by removing material from the substrate surface by friction between the probe and the substrate surface or by cutting into the substrate surface with the probe.

17. The method of any preceding claim, wherein each isolated region is substantially planar.

18. The method of any preceding claim, wherein the probe comprises a hydrophobic material.

19. The method of claim 18, wherein the probe comprises PTFE.

20. The method of any preceding claim, wherein the substrate surface is provided by an interior surface of a vessel, optionally a cell culture dish, that has undergone surface treatment to make the surface uniformly hydrophilic over a region which contains all of the selected region to be modified.

21. The method of any preceding claim, wherein the first liquid is aqueous.

22. A microfluidic arrangement manufactured using the method of any preceding claim.

23. An apparatus for manufacturing a microfluidic arrangement, comprising:

a substrate;

a scanning device configured to scan a probe over a substrate surface of the substrate;

a dispensing system configured to output liquid from a port onto the substrate surface; and a controller configured to control the scanning device such that the probe is moved over a selected region of the substrate surface while maintaining contact between the probe and the substrate surface, wherein:

the probe,the substrate surface, and the scanning are configured such that a selected region of the substrate surface is modified by the moving of the probe over the selected region, wherein the modification is such that, if a first liquid were provided over the modified selected region, an equilibrium contact angle of the first liquid with respect to contact with the modified selected region would be higher than without the modification;

the modified selected region forms one or more isolated regions on the substrate surface by surrounding each isolated region, the modified selected region forming a closed path around each isolated region;

the controller is further configured to control the dispensing system to form a microfluidic arrangement comprising a body of the first liquid in direct contact with each isolated region, and a second liquid in direct contact with, and covering, each body of first liquid and the modified selected region of the substrate surface, wherein the second liquid is immiscible with the first liquid, and each body of first liquid is held in shape predominantly by surface tension and substantially fills each isolated region.