Enzyme detection biosensors

• US 6,348,319 B1
• Filed: 11/10/1998
• Issued: 02/19/2002
• Est. Priority Date: 02/08/1996
• Status: Expired due to Fees

• Current Assignee: Biosensor Enterprises, LLC
• Original Assignee: Australian Membrane and Biotechnology Research Institute, University of Sydney
• Inventors: Braach-Maksvytis, Vijoleta Lucija Bronislava, Thomson, David Geoffrey, Raguse, Burkhard, Cornell, Bruce Andrew
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US09/125,068
• Time in Patent Office: 1,197 Days
• Field of Search: 204/400, 204/403, 204/418, 422/82.01, 422/82.02, 435/7.4, 435/7.1, 435/287.1, 435/287.2, 435/817, 436/528, 436/149, 436/806
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property

Abstract

The present invention provides a biosensor for use in detecting the presence of an enzyme or enzymes in a sample. The biosensor comprises a membrane and means for determining the impedance of the membrane. The membrane includes ionophores therein to which are attached linkers. The linkers are cleavable by the enzyme or enzymes to be detected, with the cleavage of the linker causing a change in the ability of ions to pass through the membrane via the ionophores.

Claims

1. A biosensor for use in detecting the presence of an enzyme or enzymes in a sample, the biosensor comprising a membrane and means for determining the impedance of the membrane positioned relative to the membrane such that the impedance of the membrane may be determined, the membrane having ionophores therein to which are attached linkers, the linkers being cleavable by the enzyme or enzymes to be detected, the cleavage of the linker causing a change in the ability of ions to pass through the membrane via the ionophores.

2. A biosensor as claimed in claim 1 in which the linker is attached to the membrane such that the ionophore is prevented from diffusing laterally within the membrane.

3. A biosensor as claimed in claim 2 in which the linker is attached to membrane spanning components provided in the membrane.

4. A biosensor as claimed in claim 3 in which the linker is attached to the membrane spanning component via a ligand binding pair.

5. A biosensor as claimed in claim 1 in which the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules, a plurality of ionophores and a plurality of membrane-spanning lipids prevented from lateral diffusion in the membrane, the ionophores comprising first and second half membrane spanning monomers, 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 first half membrane spanning monomers being prevented from lateral diffusion in the first layer, the second half membrane spanning monomers being linked to the membrane spanning lipids via the linker.

6. A biosensor as claimed in claim 1 in which the ionophores are gramicidin or analogues thereof.

7. A biosensor as claimed in claim 1 in which the enzyme to be detected is a protease.

8. A biosensor as claimed in claim 7 in which the protease is Prostate Specific Antigen.

9. A biosensor as claimed in claim 1 in which the enzyme to be detected is a nuclease.

24. A method of detecting the presence of an enzyme in a sample comprising adding the sample to the biosensor as claimed in claim 1 and measuring the change in impedance of the membrane.

25. A method as claimed in claim 24 in which the enzymes to be detected are proteases.

26. A method as claimed in claim 25 in which the protease is Prostate Specific Antigen.

10. A biosensor for use in detecting the presence of an enzyme in a sample, the biosensor comprising a membrane and means for determining the impedance of the membrane positioned relative to the membrane such that the impedance of the membrane may be determined, the membrane having a plurality of ionophores and a plurality of membrane-spanning components therein, the membrane-spanning components having attached thereto linker molecules to which are connected the ionophores, the linker molecules being cleavable by the enzyme to be detected, the cleavage of the linker molecules causing a change in the ability of ions to pass through the membrane via the ionophores.

11. A biosensor as claimed in claim 10 in which the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules and the membrane-spanning components are prevented from lateral diffusion in the membrane.

12. A biosensor as claimed in claim 10 in which the ionophores comprise first and second half membrane spanning monomers, 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 with the first half membrane spanning monomers being prevented from lateral diffusion in the first layer.

13. A biosensor as claimed in claim 10 in which the ionophores are gramicidin or analogues thereof.

14. A biosensor as claimed in claim 10 in which the enzyme to be detected is a protease.

15. A biosensor as claimed in claim 14 in which the protease is Prostate Specific Antigen.

16. A biosensor as claimed in claim 10 in which the enzyme to be detected is a protease.

17. A biosensor for the detection of enzymes comprising first and second zones, means to allow addition of a sample suspected to contain a protease to the first zone, the first zone containing a probe linked to a carrier via a linker cleavable by the enzyme and means to allow passage of unlinked probe from the first zone to the second zone;

the second zone including a membrane the impedance of which is dependent on the presence or absence of the probe and means to measure the impedance of the membrane positioned relative to the membrane such that the impedance of the membrane may be determined.

18. A biosensor as claimed in claim 17 in which the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules and a plurality of ionophores comprising first and second half membrane spanning monomers, 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, and a ligand provided on at least the second half membrane spanning monomers, said ligand being reactive with the probe or a portion thereof, the binding of the probe to the ligand 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.

19. A biosensor as claimed in claim 17 in which the enzymes to be detected are proteases.

20. A biosensor as claimed in claim 19 in which the protease is Prostate Specific Antigen.

21. A biosensor as claimed in claim 17 in which the enzyme to be detected is a nuclease.

22. A biosensor as claimed in claim 17 in which the half membrane spanning monomers are gramicidin or analogues thereof.

23. A biosensor as claimed in claim 17 in which the probe includes an ionphore.

27. A biosensor for use in detecting the presence of an enzyme or enzymes in a sample, the biosensor comprising a membrane and a detector positioned relative to the membrane for determining the impedance of the membrane, the membrane having ionophores therein to which are attached linkers, the linkers being cleavable by the enzyme or enzymes to be detected, the cleavage of a linker causing a change in the ability of ions to pass through the membrane via the ionophores.