FR2754469A1 - Tool for transport of liquid drops in spaced array - Google Patents

Abstract

A tool transports drops of liquid in a predetermined spatial array. The tool comprises a wire (1) mounted on a frame (3). The wire has areas which are wettable by the liquid and arranged in the predetermined array. The wettable areas are separated by non-wettable sections.

Description

 The present invention relates to a tool for transporting drops of liquid and to a process for transferring such drops, to or from a network of miniature wells capable of receiving them.

 To carry out tests or cultures on biological molecules or cultures of eukaryotic or prokaryotic cells, plates molded in thermoplastic materials such as polycarbonate or polystyrene are commonly used today, these plates comprising 96 cavities or clean wells to receive, for example, cells to be cultivated and their nutritive medium. Typically, the plate is rectangular, approximately 80 × 125 mm in size and the wells have a diameter of approximately 8 mm. These dimensions are standardized so as to allow automatic handling of the plates in devices such as a liquid handling robot, a plate reader in spectrophotometry, in fluorimetry, or in chemiluminescence, or an observation microscope. The wells are filled using sets of pipettes moved manually, which is long and tedious, or automatically. Samples of the products formed in the wells are taken, for example, using sets of stainless steel needles or plastic tips which are soaked in the wells.

 It is now envisaged to use plates with larger numbers of wells per plate, for example 384 and 1536 wells, and even plates that can have ten thousand miniature wells, or microwells, per square centimeter of surface area. the plate, as described in French patent application no. 95 13878 filed on November 22, 1995 by the applicant. In the latter case, the wells, with a diameter of the order of 20 to 50 μm and a depth of 15 to 30 μm, are distributed with a pitch of the order of 100 μm. It is understandable that such dimensions make it practically impossible to load the wells with sets of pipettes, or withdrawals from the wells with sets of metal fingers, due to the extreme miniaturization necessary for these mechanical members, impossible to achieve.

 The present invention therefore aims to provide a tool for transporting drops of liquid to or from a network of microwells distributed with a very small pitch making it impossible to use the conventional means of transport mentioned above.

 The present invention also aims to produce such a tool for simultaneously or automatically loading or unloading at least part of the wells of the network.

 Another object of the present invention is to provide a method for transferring a plurality of drops of liquid between microwells having a predetermined spatial distribution and such a tool for transporting said drops.

 These objects of the invention are achieved, as well as others which will appear on reading the description which follows, with a tool for transporting drops of liquid arranged according to a predetermined spatial distribution, this tool being remarkable in that it comprises at least one filiform support mounted on a frame releasing this support, said support having a plurality of areas wettable by said liquid, distributed in a predetermined distribution over its length, these wettable areas being separated from each other by non-wettable sections of said support.

 As will be seen below, all the wettable areas can be loaded or unloaded in drops of liquid simultaneously, which ensures very short liquid transfer operations. The pitch of the wettable areas can be very small, of the same order as that of the microwells on the plates mentioned above.

 According to one embodiment of the tool according to the invention, the frame of the tool is rectangular, the support is threadlike and stretched over this frame along successive parallel segments connected end to end, the wettable areas then being arranged in a regular two-dimensional flat distribution.

 According to another embodiment of the tool according to the invention, the areas intended for removing or depositing drops are defined by the crossing of two segments of the filiform support so as to ensure the wettability of the support at the only crossings, by capillarity. The invention also provides a method for transferring a plurality of drops of liquid between wells having a predetermined spatial distribution and a tool for transporting said drops, this method being remarkable in that a) a tool for transporting drops is produced. according to the invention, comprising a distribution of wettable areas in accordance with said distribution of wells, and b) the distribution of wettable areas of said tool and the distribution of wells are superimposed in a proximity relationship causing the deferral of at least one of the drops carried by one of the distributions in the other distribution.

Other characteristics and advantages of the present invention will appear on reading the description which follows and on examining the appended drawing in which
FIG. 1 is a diagram of an embodiment of the filiform support of the tool according to the present invention,
FIGS. 2A and 2B illustrate the method according to the invention, of loading drops of liquid on a transport tool equipped with the filiform support of FIG. 1,
FIG. 3 is an enlarged diagram which explains the mechanism for the transfer of a distribution of drops carried by the tool in FIGS. 2A and 2B, in a distribution of microwells,
FIGS. 4A and 4B illustrate the process according to the invention, applied to the transfer of a set of drops taken from a plate of microwells loaded with liquid,
FIG. 5 is a schematic perspective view of an apparatus for loading drops of different liquids onto a transport tool according to the invention,
FIG. 6 is a diagram of an element of the apparatus of FIG. 5, useful for the description of the operation of this apparatus,
FIG. 7 is a schematic perspective view of a drop transport tool according to the invention, adapted to cooperate with a two-dimensional network of microwells,
FIGS. 8A and 8B are schematic views illustrating the operation of another embodiment of the drop transport tool according to the invention,
FIG. 9 is a schematic perspective view of a two-dimensional version of the tool illustrated in FIGS. 8A and 8B,
FIG. 10 is a plan view of yet another embodiment of the tool according to the present invention,
FIG. 11 represents a detail of a variant of the tool of FIG. 9, and
FIG. 12 is a detailed view of a variant of the tool of FIG. 10.

 Referring to Figure 1 of the accompanying drawing in which there is shown schematically a filiform support 1 carrying drops 21, 22, etc ... of a liquid, distributed over the length of the support. According to an embodiment of the invention very diagrammatically shown in FIG. 3, this filiform support, or wire, 1 is mounted on a frame 3 in the form of an arc which stretches the wire between its ends.

 According to the present invention, the surface of the wire 3 is treated so that it can only carry drops of a liquid in predetermined locations, distributed along the length of the wire.

 Thus, when the wire 1 is immersed in a liquid 4 (see FIG. 2A) filling a groove with a reservoir 5 for example, and then the wire is removed from the groove (see FIG. 2B), liquid remains on the wire in the form of drops 21, 22, 23, etc. present at the determined locations or ranges, two successive drops being separated by a length or "section of wire" which is not wet.

 As an illustrative and nonlimiting example, such a wire 1 can be produced by covering an INCONEL alloy wire 20 μm in diameter, with a very hydrophobic silane overlayer, then by exposing this layer through a mask to destroy to ultraviolet radiation this layer on tracks 61, 62, 63, etc. (see FIG. 1) which must become hydrophilic again in order to be able to withstand drops 2i of a liquid. It was thus possible to carry over the course of the drops of a very small volume, from 100 to 150 picoliters.

 As a variant, the yarn 1 can carry a hydrophilic coating selectively removed outside the ranges 6i which must carry drops. More generally, the surface of the wire is treated so as to ensure the wetting of predetermined ranges thereof with a given liquid, and to prevent the wetting of the wire between these ranges.

 The assembly (1,3) constitutes a tool for transporting drops of liquid distributed over the wire 1 according to a predetermined distribution defined by the mask used during the exposure of the silane layer to ultraviolet radiation.

 If we then approach the wire 1 of the surface of a line of microwells 7i, 7i + 1, etc. ... dug into the surface of a plate 8 (see FIG. 3) of the type described in the patent application French No. 95 13878 mentioned above, the distribution of the wells on the plate being identical to that of the drops on the wire, it is understood that each of the drops 2i, 2i + 1, etc. can be placed opposite a well 7i, 7i + 1, etc ... by the use of adequate tracking means. Preferably, the spaces separating the bottoms of the wells receive a hydrophobic product 9.

The drops 2i are then sucked into the bottoms of the wells 6i and are carried over completely into them.

 We now understand that the transport tool (1, 3) according to the invention makes it possible to simply and simultaneously load a plurality of miniature wells, or microwells, 7i with a liquid, even if the pitch of the wells is very reduced as this is the case for that of the plates described in the aforementioned French patent application (of the order of 100 μm). It is clear that conventional loading methods, using micropipettes for example, use instruments that are too bulky to allow simultaneous loading of such microwells, a problem which is however completely resolved with the aid of the transport tool and the transfer according to the invention.

 This thus finds application, in particular, to cell cultures and biological assays practiced using microwell plates, cultures and tests which require in particular inputs of nutritive liquid or reagent in the microwells. Such applications are, for example, enzyme-linked immunosorbent assays (ELISA), nucleic acid hybridization, virus titration, cloning and colony screening for protein production.

 Reference is now made to FIGS. 4A and 4B to explain how the transport tool according to the invention also makes it possible to extract drops of liquid from microwell networks. In FIG. 4A, a line of microwells 7i of the plate 8 is shown diagrammatically, these wells being filled with drops 2i of a liquid from which samples have to be extracted. This is the case, for example, in cell cultures, when it is necessary to analyze products expressed or excreted in each microwell by living cells cultivated in these wells, for example for the production of monoclonal antibodies from hybridomas, for the expression of recombinant, inducible proteins or viruses from various biological systems (bacteria, yeasts, animal or plant cells).

 To do this, hydrophilic pads 6i of the perfectly dry wire 1 are placed opposite and in the vicinity of the wells 7i, as shown in FIG. 4A. As soon as the areas 6i soak in the menisci of the drops 2i projecting from the outlet of the wells on the plate 8, a part 2′i of these drops relates to the hydrophilic areas 6i opposite the wire 1, as shown in FIG. 4B. These samples 2li of the content of the wells can be transferred to microwells of other plates of such microwells for example, to constitute "imprint" plates used in microbiology.

 So far, the loading of the transport tool according to the invention has been described with droplets of the same liquid. In biological analysis in particular, it may be necessary to load adjacent wells with different liquids. In order to transfer such droplets of liquid to the tool, the invention provides the apparatus shown in FIG. 5. This comprises a rectilinear arrangement of capillary tubes 10i stretched to reach a diameter of the order of 100 μm for example, allowing them to be placed in line with a pitch identical to that of the hydrophilic areas 6i of the wire 1 of the support according to the invention.

 As shown diagrammatically in FIG. 6, an optical fiber 11i is associated with each capillary tube 10i, a microlens focusing the light leaving the fiber in the liquid filling the tube. It is known that the emission of a light pulse in the fiber causes the formation, at the open end or outlet 12i of the law tube, of a droplet of the liquid. If, as shown in FIG. 5, the wire 1 of the transport tool according to the invention is placed facing and in the vicinity of the outlets 12i of the tubes, the hydrophilic areas of this wire being located with the outlets 12i, light pulses sent into predetermined fibers lli, control the transfer onto the wire 1 of predetermined drops 2i. The capillary tubes being able to be supplied with different liquids, it is understood that the drops 2i carried by the wire 1 can have different compositions. It is then possible to transfer these drops using the tool according to the invention, and in accordance with the mechanism described in connection with FIG. 3, in different wells of a microwell plate. Such a transfer is useful in biological analysis (or in biology). An important application is the screening of synthetic or natural molecules, for the development of new drugs. Another example is diagnostic tests in biological analysis.

 It is clear that the drop transfers described above require precise identification of the wells with the hydrophilic areas of the wire 1 of the tool. Many known means for tracking can be used for this purpose, including means of the type used in photomechanical printing for tracking of multiple color selections. These means consist of rods (not shown) carried by one of the members (the tool or the plate) and penetrating into corresponding holes (not shown) dug in the other to arrange the two members in a very relative arrangement. precise.

 Hitherto, means have been described for transferring microdrops between a transport tool and only one row of microwells. The microwell plates described in the aforementioned patent application comprises a plurality of adjacent rows of microwells. Thanks to the embodiment of the transport tool according to the invention, which will now be described in connection with FIG. 7, it is possible to carry out simultaneous transfers of drops between this tool and several rows of microwells.

 In FIG. 7, the tool shown comprises a hollow frame 3 ′ which is substantially rectangular and furnished with pins 13i on two opposite edges, these pins defining support points for a wire 1 ′ passed around them so as to follow a planar trajectory in slots made up of parallel segments connected end to end.

These segments are spaced like the rows of microwells of a plate 8 comprising a regular two-dimensional distribution of such microwells. As for the tool of FIG. 2B, the wire 1 ′ carries hydrophilic areas having the same spatial distribution as the microwells of the plate 8, the rest of the surface of the wire being hydrophobic.

 It will be understood on examining FIG. 7 that the tool shown allows, after a precise tracking with the rows of wells of the plate 8, obtained for example using rods and holes (not shown), a bidirectional transfer of drops 2i7 between all of the parallel segments of the wire 1 'and all of the rows of microwells on the plate. This conveniently multiplies the number of drops of liquid carried over or removed.

 The necessary selective wettability of the surface of the wire can be obtained, as seen above, by a local modification of this surface characteristic of the wire so that the liquid clings to the areas 6i and is repelled by the parts of the wire intermediate between these areas. Other means could be used to selectively wet the wire, for example by locally giving it a high porosity, where it is desired that liquid clings to the wire. Liquid penetrates the pores thus formed on the wire, so as to then restore, by dilution for example, samples of this liquid in another liquid, or in an analysis tool.

 In FIG. 8A, a transport tool comprising a 1 "thread mounted on a frame 3 is very schematically represented. To locally increase the wettability of the thread, the latter is crossed with other tangent threads or segments of thread t 1 "2, l" 2, Irr3r etc .... at the level of the areas to be made wettable.

 Where two wires cross, the drop sampling is favored by the capillarity phenomenon.

Soaked in a liquid, or in the drops of a liquid contained in the microwells 7i of a row of such microwells, the intersections 14i of the wires 1 "and l" i are each charged with a drop 2i, as shown schematically in the Figure 8B, the intervals between drops on the wire remaining dry.

 FIG. 9 represents another embodiment of the tool according to the invention, which applies the concept developed in conjunction with FIGS. 8A and 8B. In FIG. 9, the tool shown comprises at least one wire 1 "mounted on a frame 3" using pins 13i, as for the embodiment of FIG. 7. However, here, the tensioned wire segments between two pins intersect according to two orthogonal networks whose intersections trap by capillarity of the drops 2ij to be transferred. These can then be transferred simultaneously into the microwells of a plate 8 of such microwells, or be taken from said microwells as seen above.

 It will be noted that the 1 "wire does not require the application of any local surface treatment, which is often expensive.

 It now appears that the present invention does achieve the goals set, namely to provide means for simultaneously transferring a large number of microdrops of liquid to and from a dense network of microwells inaccessible by conventional means used with much less networks. dense prior art.

 The present invention has many other advantages. It allows reproducible drops of liquid to be transferred with regard to the volume of the drops. It provides total visibility of transfer operations thanks to the fact that the transport tool is substantially transparent for an observer who performs the transfer, for example under observation under a microscope.

The tool's wire network is flexible and can be adapted to any flatness defect in the microwell plate. The tool is inexpensive to manufacture and can be discarded after a single use, which is advantageous in biology (or biological analysis). The living cells placed at the bottom of the wells stay away from the wire of the tool and cannot be damaged by it. The tracking of the tool and the microwell array can be helped and facilitated by tracking pins and, in the case of the two-dimensional tools of FIGS. 7 and 9, by the use of a moiré effect. Finally, the invention allows a collection of samples by gel sampling or a deposit of samples on or in a gel layer.

 Of course, the invention is not limited to the embodiments described and shown which have been given only by way of example. This is how the network of filiform supports of the tool according to the invention can be produced by chemical etching of a metal sheet 14 as illustrated in FIG. 10, where this engraving makes it possible to cut out a network of filiform supports. 15ij arranged like those of FIG. 9.

Incidentally, FIG. 10 shows holes 16i used to put the tool 14 in registration with a microwell plate, thanks to pins integral with this plate and passing through these holes, as mentioned above.

 As a variant, the tool of FIG. 9 could comprise a double grid of wires such as 171, 172 (see FIG. 11) these double wires defining at the intersections of the grid spaces suitable for retaining drops of larger volume. By an engraving similar to that described in connection with the tool of Figure 10, one could form, at the intersections of the filiform supports thereof, circular cutouts such as that (18) shown in Figure 12, suitable for increasing the capillary effect where the tool has to withstand drops. Also the invention is not limited to transport of microdrops from or to microwells distributed with a pitch of the order of 100 μm. It obviously extends to larger wells, distributed with a larger pitch, for example a few millimeters. Larger drops will then be obtained with wires of larger diameters, for example from 100 to 200 μm.

Claims (17)

 1. Tool for transporting drops of liquid arranged according to a predetermined spatial distribution, characterized in that it comprises at least one filiform support (l; î '; l ", l") mounted on a frame (3; 3'; 3 ") releasing this support, said support having a plurality of wettable areas (6i) by said liquid, distributed in a predetermined distribution over its length, these wettable areas (6i) being separated from each other by non-wettable sections of said support .  2. Tool according to claim 1, characterized in that the support (1; 1 ') consists of a metal wire covered with a coating not wettable by said liquid, said coating being removed at the location of the beaches (6i ) to wet with said liquid.  3. Tool according to claim 1, characterized in that the support (1; 1 ') consists of a metal wire covered with a coating wettable by said liquid, said coating being removed outside the ranges to be wetted with said liquid .  4. Tool according to claim 2, characterized in that the metal wire is made of Inconel alloy and in that the coating is a silane.  5. Tool according to claim 1, characterized in that the wettable areas (6i) are constituted by porous areas of the surface of the support (1; 1 '), separated by non-porous sections of the support.  6. Tool according to any one of claims 1 to 5, characterized in that the frame (3 '; 3 ") is rectangular, the filiform support (1'1") being stretched on this frame along successive parallel segments connected end to end, the wettable areas (6i) then being arranged in a regular two-dimensional flat distribution.  7. Tool according to claim 1, characterized in that the wettable areas (6i) are defined by the crossing of two segments (1 ", l" i) of the filiform support, clean, by capillary action, to retain drops liquid only at crossings.  8. Tool according to claim 7, characterized in that it comprises two orthogonal networks of filiform support segments, superimposed substantially in the same plane and defining by their intersections a regular two-dimensional distribution of wettable areas.  9. Tool according to claim 8, characterized in that said orthogonal networks of segments are cut from the same metal sheet (14), by chemical etching.  10. Tool according to claim 8, characterized in that the crossings of two networks are each pierced with an opening (18).  11. Tool according to claim 8, characterized in that each of said segments comprises two parallel and spaced filiform supports (171, 172).  12. Tool according to any one of the preceding claims, characterized in that the wettable areas are distributed with a pitch of the order of about 100 µm.  13. Method for transferring a plurality of drops (2i) of liquid between wells (7i) having a predetermined spatial distribution and a tool for transporting said drops, characterized in that  a) a drop transport tool is produced in accordance with any one of claims 1 to 11, comprising a distribution of wettable areas (6i) in accordance with said distribution of wells, and  b) the distribution of wettable areas (6i) of said tool and the distribution of wells (7i) are superimposed in a close relationship causing the transfer of at least one of the drops carried by one of the distributions in the other distribution.  14. Method according to claim 13, applied to the transfer of a distribution of drops carried by the transport tool (1,3) to a conformal distribution of wells, characterized in that, for loading the distribution of drops on said tool, the entire filiform support (3) is dipped in a tank (5) of the liquid to be transported.  15. The method of claim 13, applied to the transfer of a distribution of drops comprising drops of at least two different liquids, to a conformal distribution of wells, characterized in that the various wettable areas are loaded (6i) of the tool by placing them opposite aligned outlets (12i) of capillary tubes (10i), at the pitch of said areas, each tube (10i) being filled with a predetermined liquid, and the appearance of selectively is activated. drops at the outlet of said tubes, to wet said areas.  16. Method according to claim 13, applied to the sampling of fractions of liquids contained in a well distribution, characterized in that the wettable areas (6i) of the transport tool are located in register with wells (7i) well distribution correspondents, so as to wet said areas with the liquids contained in said wells.  17. Apparatus for loading drops on a tool according to any one of claims 1 to 12, characterized in that it comprises at least one rectilinear arrangement of capillary tubes (lOi) each associated with means (lli) for controlling selectively the formation of a drop of the liquid contained in the tube, at the outlet (12i) of said tube, said outlets being arranged in step with the wettable areas (6i) of the filiform support of the tool.