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Title:
IMPROVEMENTS IN CRYSTALLISATION
Document Type and Number:
WIPO Patent Application WO/2011/110812
Kind Code:
A2
Abstract:
A sitting-drop microwell plate (10, 10A) has wells (11) for crystal-forming chemicals and adjacent to each well (11) a shelf-like depression (12, 12A) on which a biological or protein specimen will be located for the formation of a crystal by exposure to vapour from the chemicals. In accordance with the invention a carrier member in the form of a sheet or film (14) of a light-transmissive material or a capillary tube (19) is located on the base (20, 20A) of each depression (12, 12A) so that a crystal will form on or in the carrier member (14,19) and can thus be removed from the microwell plate (10, 10A) for analysis without handling the crystal.

Inventors:
REARDON, Paul (Pinetrees Cottage, 24 Ellesmere RoadWeybridge, Surrey KT13 0HN, GB)
Application Number:
GB2011/000334
Publication Date:
September 15, 2011
Filing Date:
March 09, 2011
Export Citation:
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Assignee:
SWISSCI A.G. (Neuhofstrasse 74, Newheim, Zug, CH-6345, CH)
REARDON, Paul (Pinetrees Cottage, 24 Ellesmere RoadWeybridge, Surrey KT13 0HN, GB)
Attorney, Agent or Firm:
LAMBERT, Peter, Richard (Motts Barn, 103A High StreetIbstock, Leicestershire LE67 6LJ, GB)
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Claims:
CLAIMS:

1. Crystallisation apparatus comprising a sitting-drop crystallisation plate (10,1 OA) formed with mutually separated wells (11) each to contain a solution of chemicals designed to promote the growth of crystals and adjacent and opening to each well at least one relatively shallower depression (12.12A) having a shelf-like base (20,20A) and a wall (21,21 A) on three sides to receive a biological or protein specimen characterised by the provision of a carrier member (14,19) removably positioned on and contiguous with the base (20,20 A) of a said depression (12, 12 A) whereby a crystal may be formed on or in the carrier member (14,19) and subsequently removed from the plate (10,10A) by removing the carrier member (14,19).

2. Apparatus as claimed in claim 1 characterised in that said carrier member (14) is a sheet or film of a light-transmissive material substantially co-extensive with the base (20) of the associated depression (12)

3. Apparatus as claimed in claim 2 characterised in that each carrier member (14) is retained on the base (20) of the associated depression (12) by being coated on one side with a low tack adhesive.

4. Apparatus as claimed in claim 2 characterised in that each carrier member (14) is retained on the base of the associated depression (12) by the application of heat. 5. Apparatus as claimed in any one of claims 2 - 4 characterised in that each carrier member (14) is formed with a tab (15) which will extend beyond the base of the associated depression (12) to assist manipulation of the carrier member (14).

6. Apparatus as claimed in claim 1 characterised in that the carrier member is a capillary tube (19).

7. Apparatus as claimed in claim 6 characterised in that the capillary tube (19) removably locates in a groove (18) in the base (20A) of the associated depression (12A).

8. Apparatus as claimed in claim 7 characterised in that the base (20 A) of each depression (12A) has a concave area (16) to receive the biological or protein specimen separated from the associated well (11) by a raised area (17), said groove (18) extending across the raised area (17) from the concave area (16) to the well (11) whereby when a capillary tube (19) is located in the groove (18) the tube (19) will open at one end to the concave area (16) and at its other end to the well (11).

Description:
IMPROVEMENTS IN CRYSTALLISATION

This invention relates to improvements in crystallisation and more specifically to improvements in the techniques whereby biological or protein specimens are exposed to vapour from crystal-forming solutions, the crystals then being subjected to analysis for example by x-ray.

One type of apparatus currently available uses a so-called "sitting-drop microwell plate" in which, typically, 96 wells are formed to contain the crystal-forming solutions. Biological specimens are located on shelf-like, shallower depressions adjacent the wells and the wells and associated depressions are isolated from one another by a cover for the plate. After exposure of the specimens to vapour from the wells for a given time crystals will have formed on some of the depressions. The crystals are identified using a microscope and are removed from the microwell plate using a pipette or a so-called loop to be subjected to analysis, for example by x-ray. Such a sitting-drop microwell plate is disclosed in British Patent Application Serial No. 2427688 and in the corresponding U.S. Patent Application Serial No. 2006/0292654. Reference is made to these publications for a clearer understanding of the present invention. The crystals are very fragile and short-lived. Any handling with a pipette or loop is likely to destroy them and great care has to be exercised. A principal object of the present invention is to minimise or eliminate handling of the crystals by forming them on or in carrier members which are removable from the microwell plates. If these carrier members are of light-transmissive material their presence together with the crystals will not interfere with subsequent x-ray or other analysis of the crystals.

In accordance with the present invention there is provided crystallisation apparatus comprising a sitting-drop crystallisation plate formed with mutually separated wells each to contain a solution of chemicals designed to promote the growth of crystals and adjacent and opening to each well at least one relatively shallower depression having a shelf-like base and a wall on three sides to receive a biological or protein specimen characterised by the provision of a carrier member removably positioned on and contiguous with the base of a said depression whereby a crystal may be formed on or in the carrier member and subsequently removed from the plate by removing the carrier member. In one embodiment of the present invention said carrier member is a sheet or film of a light- transmissive material substantially co-extensive with the base of the associated depression

Each carrier member may be retained on the base of the associated depression by being coated on one side with a low tack adhesive. Alternatively each carrier member may be retained on the base of the associated depression by the application of heat.

Each carrier member may be formed with a tab which will extend beyond the base of the associated depression to assist manipulation of the carrier member. In another embodiment of the present invention the carrier member is a capillary tube.

The capillary tube may be removably located in a groove in the base of the associated depression. The base of each depression may have a concave area to receive the biological or protein specimen separated from the associated well by a raised area, said groove extending across the raised area from the concave area to the well whereby when a capillary tube is located in the groove the tube will open at one end to the concave area and at its other end to the well.

Preferred embodiments of the invention will now be described by way of non-limitative example with reference to the accompanying drawings, in which:

Figure 1 is a perspective view from above of a first embodiment of a sitting-drop microwell plate;

Figure 1 A is an enlarged detail of one of the wells of the plate of Figure 1 ; Figure 2 is a sectional elevation taken on the line Η-Π of Figure 1,

Figure 2A is an enlarged detail of one of the wells and associated depressions of the plate as shown in Figure 2;

Figure 3 is a perspective view on an enlarged scale of one of the carrier members used with the microwell plate of Figures 1 and 2, and

Figures 4 and 5 are perspective views from above on an enlarged scale similar to Figure 1 A of one of the wells of a modified microwell plate.

The sitting-drop microwell plate 10 of Figures 1, 1A, 2 and 2 A has 96 wells indicated at 11 to contain crystal-forming solutions. Adjacent and opening to each well 11 is a relatively shallower depression 12 (best seen in Figure 2 A) providing a shelf-like location for biological or protein samples which are to be exposed to vapour from the wells 11. Each depression has a generally flat base 20 with a wall 21 on three sides. A cover (not shown) is used to cover the plate 10 during crystal formation and cooperates with lands 13 between the wells 11 to isolate each well 11 and its associated depression 12 from adjacent wells and depressions.

In a modification (not shown) more than one depression 12 may be associated with each well 11.

In accordance with a first embodiment of the present invention there is located in each depression 12 a carrier member 14 consisting of a thin film, sliver or sheet of a light- transmissive material, such as a suitable plastics material or glass, shaped to fit the depression, contiguous and generally coextensive with the base 20 of the depression.

Optionally the carrier member 14 is formed with a tab 15 which, in use, extends out of the depression 12 and into the associated well 11 where it can be easily grasped with tweezers. These tiny carrier members 14 may be kept in position in the depressions 12 by being coated on one side with a weak adhesive, known as a "low tack" adhesive. Alternatively they may be kept in position by a heat treatment, but in that event it will be necessary to use a scalpel to remove them and the tab 15 will be unnecessary. By this arrangement crystals are formed not directly on the microwell plate 10 but on the carrier members 14 and may therefore be removed for analysis without touching them simply by removing the carrier members. The second embodiment of the present invention illustrated in Figures 4 and 5 utilises a sitting-drop microwell plate 10A similar to the plate 10 of Figures 1 and 2 in that it has 96 wells 11 but the associated, relatively shallower shelf-like depressions 12A instead of having completely flat bases have respective concavities 16 and, extending across the raised area 17 between the concavity 16 and the well 11, a groove 18 in which a capillary tube 19 is removably located. This capillary tube 19 is of a light-transmissive material such as glass. It may be a glass tube coated with a light-transmissive plastics material.

In use of the plate 10A of Figures 4 and 5 a biological or protein specimen is placed in each concavity 16 and crystal forming chemicals are placed in each well 11. For example 100 nanolitres of a screen crystallisation agent and 100 nanolitres of a protein may be put in a concavity 16. A cover (not shown) is then fitted to the plate 10A to isolate each well 11 and its associated depression 12A from the others. The biological or protein specimen in each concavity 16 is drawn by capillary action into one end of the associated tube 19 and vapour from the chemicals in the associated well 11 is drawn into the other end of the tube. In the course of time crystals will form in at least some of the capillary tubes 19, which can then be removed from their grooves 18 and taken for analysis, for example by x-ray. This procedure eliminates having to handle the delicate crystals.