WO2004029338A1

WO2004029338A1 - Dialysis apparatus

Info

Publication number: WO2004029338A1
Application number: PCT/GB2002/003717
Authority: WO
Inventors: Stephen Lee
Original assignee: Rayner, Paul
Priority date: 2001-08-10
Filing date: 2002-08-08
Publication date: 2004-04-08
Also published as: AU2002326011A1

Abstract

An apparatus suitable for carrying out microdialysis is described which comprises a chamber (1) for holding a sample, disposed in a housing, the external surfaces of which are shaped to secure a dialysis membrane (5) across the mouth of the chamber (1). The housing includes a handle (4) to allow the membrane (5), when secured, to be brought into or out of contact with a solution of different ionic strength form the sample and retention means (3) so that the membrane (5) is maintained in contact with the solution. The apparatus is useful for carrying out protein crystallization experiments.

Description

DIALYSIS APPARATUS

The invention relates to apparatus suitable for carrying out dialysis on small volume samples, usually of the microlitre order and more commonly referred to as microdialysis. The invention also relates to methods of microdialysis using the apparatus as well as to other applications of the apparatus such as for protein crystallization either by vapour diffusion or by other means.

Devices are known in the art which allow dialysis of small volumes of solution. In a typical example such a device includes a sample chamber, which may have a volume of anything from about lμl to about 300μl. The chamber is usually within a circular housing which has a circumferential groove on the external surface for receiving an o-ring, which is used to secure a dialysis membrane across the mouth of the chamber once the sample has been introduced. The device is then contacted with the solution against which the sample is to be dialysed.

Such known devices suffer from the disadvantage that they are difficult to handle, both in introduction and removal from the dialysis buffer and in the application of the membrane, where it is easy to trap air bubbles between the membrane and the sample. A further disadvantage is that if one wishes to try out a number of different dialysis conditions, several different receptacles are needed, taking up a lot of bench space. In addition, it is not possible to visualize conditions in the sample chamber while the device is in use, which may be desirable for some applications.

The present invention relates to apparatus for microdialysis which overcomes these difficulties. In a first aspect the invention relates to an apparatus suitable for carrying out microdialysis which comprises a chamber for holding a sample, said chamber being disposed in a housing, the external surfaces of which are shaped to secure a dialysis membrane across the mouth of said chamber, said apparatus being characterized in that said housing includes a handle to allow said membrane when secured to be brought into or out of contact with a solution of different ionic strength from said sample and retention means to allow said membrane to be maintained in contact with said solution.

The sample chamber may have a volume in the region of about 1 μl up to about 300μl, although the usual range would be between about 5μl and about 200μl and most preferably between from about 5μl to about 20μl. In view of the small size the provision of a handle allows for much easier manipulation of the apparatus so that improper application of, or damage to, the membrane is avoided.

In a preferred embodiment the sample chamber is cylindrical and is disposed within a housing of circular horizontal cross-section. This allows a circumferential groove to be present on the external surface of the housing so that the dialysis membrane of the appropriate molecular weight cut-off (MWC) may be fitted over the mouth of the chamber and sealed with an o-ring of elastic material such, as rubber or other elastomer. As an alternative to the o-ring a "snap-on" cap member of sufficiently flexible material, preferably a plastics material, can be used whereby said cap is provided with a circumferential lip member capable of engaging in the circumferential groove in the housing. In order that the membrane can be exposed to the solution of different ionic strength the cap member is provided with a hole, preferably circular, which, when snapped to the housing, is substantially in alignment with the mouth of the chamber. In an alternative embodiment the external surface of the housing is shaped to provide a screw thread. This allows the membrane to be secured over the mouth of the chamber by a threaded cap member. Again, the cap member is provided with a hole, preferably circular which when screwed to the housing is substantially in alignment with the mouth of the chamber.

In embodiments in which a cap member is used it is preferable that the top surface of the housing surrounding the sample chamber be flat in order to ensure proper sealing of the chamber by the membrane. In embodiments in which the membrane is sealed by means of an ^λo'ring the top of the housing may be bevelled rather than flat.

A particular advantage of the present apparatus is that it may be sized and shaped so that the sample chamber and membrane fit into a well of a receptacle comprising multiple cylindrical wells and the retention means permits the membrane to be held at an optimum distance from the bottom of the well for efficient dialysis. A multiwell receptacle allows multiple samples to be processed simultaneously while minimizing the amount of space required. Particularly preferred are 24 well tissue culture plates such as the known VDX or Linbro plates. These can be obtained from Hampton Research, 27632, El Lazo Road, Suite 100, Laguna Niguel, CA 92677-3913, USA.

Preferred retention means to hold the chamber and housing in the correct position within the well include at least one catch member protruding from the housing, providing the apparatus with a diameter greater than the diameter of the well and positioned on the housing between the sample chamber and the handle. In a particular preferred embodiment the catch member is a circular flange capable of engaging the rim of the well. The flange may include a seal of rubber or other suitable elastomer around its circumference in order to form a seal with the well rim.

The handle may be any shape but where the housing is cylindrical it is practical for the handle to be in the form of a rod of circular cross-section. For certain applications it is desirable for the handle to have a flat surface at the end distal to the retention means while, in other cases, it may have an alternative shape as discussed below.

The apparatus of the invention may be made from any suitable material, for example mild steel or a plastics material, but preferred is a material that is substantially transparent such as a clear plastics material or glass. A particularly preferred material is perspex which may be suitably moulded. An advantage of the use of a transparent material is that it allows visualisation of the sample chamber through the handle during use. As will be discussed further below, one of the applications of the apparatus of the invention is for crystallization of proteins. By use of the transparent material it is possible to determine the presence of crystals in the sample chamber and/or their rate of growth, without removal of the apparatus from the well and hence disturbance of the crystals.

In one embodiment the handle is shaped as a lens so as to provide magnification of the sample. ^•

In a second aspect the invention provides a method of carrying out microdialysis of a sample which comprises the steps of : a) introducing a sample into the sample chamber of the apparatus of the invention as described above, b) securing a dialysis membrane across the mouth of said chamber, c) moving the apparatus so as to bring the membrane into contact with a solution of different ionic strength to the sample, said solution being contained in a receptacle, d) creating a seal between the mouth of said receptacle and the retention means of the apparatus and e) allowing dialysis to proceed.

The sample is usually placed in the chamber so as to create a slight dome of liquid at the top edge of the chamber. A dialysis membrane, chosen with the desired molecular weight cut-off, may then be secured across the mouth of the chamber. In embodiments of the invention in which the membrane is secured by means of an elastic "O-ring" , a "Golf Tee Applicator" or "CRE Applicator" may be used. A Golf Tee Applicator is available from- Hampton Research, 27632 El Lazo Road, Suite 100, Laguna Niguel, CA 92677-3913, USA.

In preferred embodiments the receptacle is a well of a multiwell plate, for example a 24 well tissue culture plate of the VDX or Linbro type, into which the sample loaded apparatus is introduced. Preferably the seal is substantially airtight to prevent evaporation of the solutions to the atmosphere. Typically this may be secured by using a grease between the rim of the well and the retention means of the apparatus, in particular between the circular flange and the well rim. Alternatively, an ^λo'ring of rubber or other elastomer may be disposed around the circular flange to engage the rim of the well or, as aforesaid the apparatus itself may be provided with a rubber lip seal around the circular flange.

The ionic strength of the dialysis buffer may be higher or lower than that of the sample but compounds of smaller size than the pore size of the membrane will diffuse across down a concentration gradient until the system reaches equilibrium.

Apart from microdialysis the apparatus of the invention is equally useful for protein crystallization.

Thus, in a third aspect the invention provides a method of producing a crystallized protein comprising the steps of: a) introducing a solution comprising the protein to be crystallized into the sample chamber of any of the embodiments of the apparatus described above, b) securing a dialysis membrane over the mouth of the chamber, c) bringing the membrane into contact with a^" protein precipitating solution, said solution being contained in a receptacle, d) creating a seal between the mouth of the receptacle and the retention means of the apparatus and e) allowing protein crystallization to take place.

In preferred embodiments the protein solution will be introduced into the sample chamber and the membrane applied as described above for the dialysis applications. Similarly it is preferred that the protein precipitating solution will be placed in the well of a 24 well tissue culture plate of the type described above.

For protein crystallization it is particularly preferred that the apparatus be made of a substantially transparent material as described above so that the formation of crystals in the sample chamber may be viewed through the handle. Preferably, the handle is shaped so as to provide magnification of the sample.

The crystals may be recovered from the sample chamber for further processing, such as for example for X-ray diffraction experiments or the apparatus may be used as a suitable means of storage of the crystals, and for heavy atom/ligand soak experiments. In yet a further aspect of the invention the apparatus of the invention may be used in an alternative method of protein crystallisation using vapour diffusion. Such use requires an embodiment of the apparatus in which the end of the handle distal to the retention means has a flat surface. The apparatus is used in an ^Λinverted' form in which the handle is introduced into the well of a receptacle with the sample chamber uppermost and exposed to the air. A "hanging drop" comprising a solution of the protein to be crystallised is first applied to the flat surface of the handle and it is then introduced into a well containing a protein precipitant solution in a manner such that there is no contact between the hanging drop and the precipitant solution. An airtight seal is created between the mouth of the well and the retention means. Water vapour from the hanging drop will, by diffusion, equilibrate with the protein precipitant solution and when protein supersaturation occurs, protein crystals will form on the flat end of the handle. Again the crystals may be removed and subject to further processing or may be maintained on the end of the handle as a form of storage device.

In a preferred embodiment the flat surface of the handle is siliconized to provide a hydrophobic , water repellant surface in case glass material is used.

Preferred embodiments of the invention will now be described with reference to the accompanying drawings in which: Figure 1 shows a cross-sectional view of an o- ring,

Figure 2 shows a cross-sectional view of a dialysis membrane disk,

Figure 3 shows a cross-sectional view of one embodiment of the microdialysis apparatus in accordance with the invention,

Figure 4 shows a cross-sectional view of the whole microdialysis apparatus of Figure 3, with the dialysis membrane disk secured on top of the chamber by an o-ring.

Figure 5 shows a top view of the whole microdialysis apparatus of Figure 3,

Figure 6 shows a bottom view of the whole microdialysis apparatus of Figure 3,

Figure 7 shows a cross-sectional view of the grease ring applied on the well rim of a, 24-well tissue culture plate,

Figure 8 shows a cross-sectional view of an alternative embodiment of the apparatus in which the membrane is secured across the mouth of the chamber by a snap-on cap, Figure 9 shows a cross-sectional view of an alternative embodiment of the apparatus in which the external surface of the housing is screw-thread for receipt of the threaded cap member,

Figure 10 shows crystals of glucose isomerase prepared using the apparatus in accordance with the invention and

Figure 11 shows crystals of lyzozyme prepared using apparatus in accordance with the invention.

Referring first to Figure 3, in one embodiment the microdialysis apparatus of the invention comprises a chamber 1 for holding the sample, a circumferential groove 2 shaped for retaining an elastic* o-ring, a retention means in the form of a circumferential flange 3 and a handle portion 4. As shown in Figure 4, to use the apparatus the dialysis membrane 5 is applied across the top of the apparatus to cover the mouth of the chamber 1 and is secured to the housing by an o-ring 6.

In Figure 7 the apparatus is shown in use, inserted into the well 7 of a 24-well tissue culture plate 9. Grease 8, is applied between the circumferential flange 3 and the rim of the well 7 to create an airtight seal.

An alternative embodiment of the apparatus is shown in Figure 8 in which the o-ring 6 is replaced by a "snap-on" cap 12, said cap having a circumferential lip 14 which engages with the groove 2 in the housing. The cap 12 has a hole 14 which aligns with the mouth of the chamber 1 when the cap is snapped on to the apparatus housing.

A further alternative embodiment of the apparatus is shown in Figure 9 in which the circumferential groove 2 is replaced by screw thread 15. The dialysis membrane 5 is then secured to the apparatus by means of a screw threaded cap 10 having a hole 11 which aligns with the mouth of the chamber 1 when the cap is screwed to the apparatus housing. The embodiment is used in exactly the same manner as the previously described embodiment.

The invention will now be further exemplified with reference to the following experiments.

EXAMPLE 1 Glucose Isomerase Crystallization

The apparatus as shown in Figures 1 to 7 was used to crystallize glucose isomerase. 2.5μl of 30mg/ml Glucose Isomerase in 6mM tris at pH8.4 was added to the sample chamber and was dialysed against 500μl of 1.5M Ammonium Sulphate at pH 8.4 and room temperature. The crystals produced are shown in Figure 10.

EXAMPLE 2

The apparatus as shown in Figures 1 to 7 was used to crystallize lyzozyme. 2-5μl 50mg/ml lyzozyme solubilzed in 50 mM NaOAc at pH 7.4 was added to the sample chamber and dialysed against 500μl 4% NaCl at room temperature. The crystals produced are shown in Figure 11.

Claims

CLAIMS :

1. An apparatus suitable for carrying out microdialysis which comprises a chamber for holding a sample, said chamber being disposed in a housing, the external surfaces of which are shaped to secure a dialysis membrane across the mouth of said chamber, said apparatus being characterised in that said housing includes a handle to allow said membrane when secured to be brought into or out of contact with a solution of different ionic strength from said sample and retention means to allow said membrane to be maintained in contact with said solution.

2. An apparatus according to claim 1 wherein said chamber is cylindrical and is disposed concentrically within a housing of circular cross- section.

3. An apparatus as claimed in claim 2 wherein the external surface of said housing includes a circumferential groove via which said membrane may be secured with an o-ring.

4. An apparatus as claimed in claim 2 wherein the external surface of said housing includes a circumferential groove via which said membrane may be secured with a circular cap having a circumferential lip which engages with said groove, said cap having a circular hole which, when said cap is applied to said housing, is substantially in alignment with the mouth of said chamber.

5. An apparatus as claimed in claim 2 wherein the external surface of said housing includes a screw thread whereby said membrane my be held in place by a threaded cap member, said cap member having a circular hole which, when said cap is screwed to said housing, is substantially in alignment with the mouth of said chamber .

6. An apparatus as claimed in any one of claims 2 to 5 wherein said housing is adapted such that said chamber and membrane fit into a well of a receptacle comprising multiple cylindrical wells and said retaining means permits said membrane to be maintained at an optimal distance from the bottom of said well.

7. An apparatus as claimed in claim 6 wherein said retention means comprises a catch member protruding from said housing, said catch member providing said apparatus with a diameter greater than the diameter of said well.

8. An apparatus as claimed in claim 7 wherein said catch member is a circular flange capable of engaging the rim of the well.

9. A apparatus as claimed in any one of claims 6 to 8 wherein said receptacle is a 24 well tissue culture plate.

10. An apparatus as claimed in any one of the preceding claims which is moulded or machined from a transparent material.

11. An apparatus as claimed in claim 10 wherein said handle is in the shape of a rod of circular cross-section.

12. An apparatus as claimed in claim 10 or claim 11 wherein said handle allows visualisation of the sample in si tu .

13. An apparatus as claimed in claim 12 wherein said handle is moulded or machined to provide magnification of said sample.

14. An apparatus as claimed in any one of the preceding claims wherein said sample chamber is no less than about one and not more than about 300μl in volume.

15. An apparatus as claimed in claim 14 wherein said sample chamber is no less than about 5 and not^" more than about 20μl in volume.

16. An apparatus as claimed in any preceding claim wherein the end of the handle distal to the retention means has a flat surface.

17. A method for carrying out microdialysis of a sample comprising the steps of: a) introducing said sample into the sample chamber of an apparatus as claimed in any one of claims 1 to 16, b) securing a dialysis membrane across the mouth of said chamber, c) moving said apparatus so as to bring said membrane into contact with a solution of different ionic strength to said sample, said liquid being contained in a receptacle d) creating a seal between the mouth of said receptacle and the retention means of said apparatus and e) allowing dialysis to proceed.

18. A method as claimed in claim 17 wherein the seal that is created is substantially airtight.

19. A method as claimed in claim 17 or claim 18 wherein said membrane is brought into contact with a solution of lower ionic strength than said sample.

20. A method as claimed in claim 17 or claim 18 wherein said membrane is brought into contact with a solution of higher ionic strength than said sample.

21. A method as claimed in any one of claims 17 to 20 wherein said sample is a protein solution.

22. A method for producing a crystalized protein comprising the steps of: a) introducing a solution comprising said protein into the sample chamber of an apparatus as claimed in any one of claims 1 to 16, b) securing a dialysis membrane over the mouth of said chamber, c) bringing said membrane into contact with a protein precipitating solution, said solution being contained in a receptacle, d) creating a seal between the mouth of said receptacle and the retention means of said apparatus and e) allowing protein crystallization to take place.

23. A method as claimed in claim 22 wherein the seal that is created is substantially airtight.

24. A method as claimed in claim 22 or claim 23 which includes the additional step of recovering the crystalized protein.

25. A crystalized protein obtainable using the method as claimed in 24.

26. A method of producing a crystallised protein by vapour diffusion comprising the steps of: a) applying to the flat surface of the handle of an apparatus are claimed in 16 a drop of a first solution comprising the protein to be crystallised so as to form a hanging drop, b) inserting said handle and said hanging drop into a receptacle containing a protein precipitant solution, the volume of said precipitant solution being such that the retention means of said apparatus can prevent contact between said hanging drop and said precipitant solution, c) creating a substantially air-tight seal between the mouth of said receptacle and said retention means and d) allowing diffusion of water vapour between said hanging drop and said protein precipitant solution until crystallisation of said protein occurs.

27. A crystallised protein obtainable by the method of claim 26.

: 397173: SCB: P: LONDOCS