Receptor membranes and ionophore gating

• US 5,874,316 A
• Filed: 10/30/1996
• Issued: 02/23/1999
• Est. Priority Date: 01/27/1989
• Status: Expired due to Fees

• Current Assignee: Biosensor Enterprises, LLC
• Original Assignee: Australian Membrane and Biotechnology Research Institute
• Inventors: Cornell, Bruce A., Pace, Ronald J., Braach-Maksvytis, Vijoleta L. B., Osman, Peter D. J., Raguse, Burkhard, King, Lionel G.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Swartz, Rodney P.
• Application Number:
  US08/741,299
• Time in Patent Office: 846 Days
• Field of Search: 435/7.1, 435/7.2, 435/7.21, 435/317.1, 436/501, 436/512, 436/518, 436/524, 436/525, 436/532, 436/547, 436/806, 436/829
• US Class Current: 436/518
• CPC Class Codes: B01D 2325/48 Antimicrobial properties
• CPC Class Codes: B01D 69/02 characterised by their properties
• CPC Class Codes: B01D 69/122 Separate manufacturing of ultra-thin membranes
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

The present invention provides a membrane the conductivity of which is dependent on the presence or absence of an analyte. The membrane comprises a closely packed array of self-assembling amphiphilic molecules and two ionophore components. A receptor molecule reactive with the analyte is provided on one of the ionophore components. The binding of the analyte to the receptor molecule causes a change in the relationship between the ionophore components such that the flow of ion across the membrane is prevented or allowed. The ionophore components are preferably selected from the group consisting of amphotericin B, gramicidin A monomers and combinations thereof, with gramicidin A monomers being particularly preferred. The present invention also provides a membrane including receptors directed against the Fc region of antibodies. These receptors are preferably derived from polyclonal antibodies. These membranes provide a "generic" surface which will bind antibodies in a manner such that the antigen binding regions of the antibody are not hindered. The present invention further provides a device adapted for implantation in a mammalian body, the device being characterized in that it is coated with a membrane comprising a closely packed array of self-assembling amphiphilic molecules and receptor molecule. The receptor molecules being such that the attachment of specific cells to the membrane is enhanced or avoided. It is particularly preferred that the receptor molecules are directed against fibronectin, vitronectin, endothelial cells, or epithelial cells. It is further preferred that the membrane coating the device also includes a plurality of ion channels such as gramicidin.

Claims

1. A membrane in which the conductance of the membrane is dependent on the presence or absence of an analyte, the membrane comprising:

a first and second layer;

a closely packed array of amphiphilic molecules and a plurality of gramacidin ionophores comprising a first and second half membrane spanning monomer, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer, the second half membrane spanning monomers being capable of lateral diffusion within the second layer independent of the first half membrane spanning monomers, the first half membrane spanning monomers being prevented from lateral diffusion in the first layer;

a first receptor molecule provided on at least the second half membrane spanning monomers; and

in which a proportion of the amphiphilic molecules are membrane spanning amphiphiles which are prevented from lateral diffusion in the membrane to which are attached a second receptor molecule, the binding of an analyte to the first and second receptor molecules causing a change in the relationship between the first half membrane spanning monomers and the second half membrane spanning monomers such that the flow of ions across the membrane via the ionophores is allowed or prevented.

2. A membrane as claimed in claim 1 in which the first half membrane spanning monomers are prevented from lateral diffusion in the first layer by cross-linking the monomers and the amphiphilic molecules or by covalent attachment of the monomers to a solid surface.

3. A membrane as claimed in claim 1 in which the membrane spanning amphiphiles being archeobacterial lipids or tail to tail chemically linked bilayer amphiphiles.

4. A membrane as claimed in claim 1 in which the amphiphilic molecules are membrane spanning amphiphiles, the membrane spanning amphiphiles being archeobacterial lipids or tail to tail chemically linked bilayer amphiphiles.

5. A membrane as claimed in claim 1 in which the half membrane spanning monomers are gramicidin A monomers.

6. A membrane as claimed in claim 1 in which the first half membrane spanning monomers and the amphiphilic molecules in the first layer each include or are decorated with at least one moiety cross-linked with at least one corresponding moiety on another of these molecules.

7. A membrane as claimed in claim 1 in which the first receptor molecule has two receptor sites for the analyte.

8. A membrane as claimed in claim 1 in which the first and second receptor molecules are the same or different and are selected from the group consisting of polyclonal or monoclonal antibodies, antibody fragments, enzymes antigens, lectins, haptens, chelating agents and dyes.

9. A membrane as claimed in claim 8 in which the first and second receptor molecules are antibodies or antibody fragments.

10. A membrane as claimed in claim 1 in which the membrane is attached to a solid surface.

11. A membrane as claimed in claim 1 in which the second layer has a phase transition temperature such that cooling of the membrane below this temperature induces phase separation in the second layer thereby releasing any analyte bound to the first and/or second receptor molecules.

12. A membrane as claimed in claim 1 in which the first receptor molecule is attached to the second half membrane spanning monomer via a linker group, the linker group comprising a spacer group and one or more reactive groups, the spacer group being attached to the second half membrane spanning monomer and the reactive group providing attachment to the receptor molecule.

13. A membrane as claimed in claim 12 in which the spacer group is selected from the group consisting of hydrocarbons, oligomers of ethylene glycol, and oligo peptides;

and the reactive group is selected from the group consisting of N-hydrosuccinimide esters, esters for covalent coupling to amine groups on proteins, hydrazine derivatives for coupling onto oxidised sugar residues, maleiimide derivatives, biotin, streptavidin and antibodies.

14. A membrane as claimed in claim 1 in which the first and second receptor molecules bind to different sites on the analyte, such that binding of the first and second receptors to the analyte results in a change in the flow of ions across the membrane.

15. A membrane as claimed in claim 1 in which the first and second receptor molecules are bound to different sites on the analyte or an analog thereof, the addition of analyte effecting competitive binding with the first and second receptor molecules resulting in a change in the flow of ions across the membrane.

16. A membrane as claimed in claim 1 in which the first receptor molecule binds to the second receptor molecule.

17. A membrane in which the conductance of the membrane is dependent on the presence or absence of an analyte, the membrane comprising:

a first and second layer;

a closely packed array of amphiphilic molecules and a plurality of gramacidin ionophores comprising a first and second half membrane spanning monomer, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer, the second half membrane spanning monomers being capable of lateral diffusion within the second layer independent of the first half membrane spanning monomers, the first layer being in crystalline phase such that the first half membrane spanning monomers are prevented from lateral diffusion in the first layer; and

a first receptor molecule provided on at least the second half membrane spanning monomers, said first receptor molecule binding with the analyte or a portion thereof, the binding of the analyte to the first receptor molecule causing a change in the relationship between the first half membrane spanning monomers and the second half membrane spanning monomers such that the flow of ions across the membrane via the ionophores is allowed or prevented.

18. A membrane as claimed in claim 17 in which a proportion of the amphiphlic molecules are membrane spanning amphiphiles, the membrane spanning amphiphiles being archeobacterial lipids or tail to tail chemically linked bilayer amphiphiles.

19. A membrane as claimed in claim 17 in which the amphiphilic molecules are membrane spanning amphiphiles, the membrane spanning amphiphiles being archeobacterial lipids or tail to tail chemically linked bilayer amphiphiles.

20. A membrane as claimed in claim 17 in which the half membrane spanning monomers are gramicidin A monomers.

21. A membrane as claimed in claim 17 in which the membrane includes a plurality of second receptor molecules having receptor sites remote from the first layer.

22. A membrane as claimed in claim 21 in which the second receptor molecules are prevented from diffusing laterally within the membrane.

23. A membrane as claimed in claim 22 in which the second receptor molecules are attached to membrane spanning amphiphiles.

24. A membrane as claimed in claim 17 in which the first receptor molecule has two receptor sites for the analyte.

25. A membrane as claimed in claim 21 in which the first and second receptor molecules are the same or different and are selected from the group consisting of polyclonal or monoclonal antibodies, antibody fragments, enzymes, antigens, lectins, haptens chelating agents and dyes.

26. A membrane as claimed in claim 25 in which the first and second receptor molecules are antibodies or antibody fragments.

27. A membrane as claimed in claim 17 in whch the membrane is attached to a solid surface.

28. A membrane as claimed in claim 21 in which the second layer has a phase transition temperature such that cooling of the membrane below this temperature induces phase separation in the second layer thereby releasing any analyte bound to the first and/or second receptor molecules.

29. A membrane as claimed in claim 17 in which the first receptor molecule is attached to the second half membrane spanning monomer via a linker group, the linker group comprising a spacer group and one or more reactive groups, the spacer group being attached to the second half membrane spanning monomer and the reactive group providing attachment to the receptor molecule.

30. A membrane as claimed in claim 29 in which the spacer group is selected from the group consisting of hydrocarbons, oligomers of ethylene glycol, and oligo peptides;

and the reactive group is selected from the group consisting of N-hydrosuccinimide esters, esters for covalent coupling to amine groups on proteins, hydrazine derivatives for coupling onto oxidised sugar residues, maleiimide derivatives, biotin, streptavidin and antibodies.

31. A membrane as claimed in claim 29 in which the reactive group is biotin, biotin-streptavidin or an antibody or antibody fragment directed against the Fc portion of an antibody.

32. A membrane as claimed in claim 17 in which the first receptor molecules on separate second half membrane spanning monomers bind to different sites on the analyte, such that binding of the first receptors to the analyte results in a change of the flow of ions across the membrane.

33. A membrane as claimed in claim 17 in which the first receptor molecules on separate second half membrane spanning monomers are bound to different sites on the analyte or an analog thereof, such that the flow of ions across the membrane is prevented, the addition of analyte effecting competitive binding with the first receptor molecules resulting in the flow of ions across the membrane.