Biosensor compositions and methods of use

• US 20050208574A1
• Filed: 05/16/2005
• Published: 09/22/2005
• Est. Priority Date: 02/11/2000
• Status: Abandoned Application

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Bayley, Hagan P., Movileanu, Liviu, Howorka, Stefan G.
• Application Number:
  US11/129,917
• Publication Number:
  US 20050208574A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743
• CPC Class Codes: Y10S 530/825 Bacteria

Abstract

Provided are pore-subunit polypeptides covalently linked to one or more sensing moieties, and uses of these modified polypeptides to detect and/or measure analytes or physical characteristics within a given sample.

Claims

1. A modified pore-subunit polypeptide comprising a pore-subunit polypeptide covalently linked to at least a first sensing moiety, wherein said modified pore-subunit polypeptide assembles into an oligomeric pore assembly in the presence of a plurality of pore-subunit polypeptides.

2. The modified polypeptide of claim 1, wherein said sensing moiety is a functional group.

3. The modified polypeptide of claim 2, wherein said functional group is an analyte-binding functional group.

4. The modified polypeptide of claim 2, wherein said functional group is a synthetic molecule.

5. The modified polypeptide of claim 4, wherein said functional group is a calixarene or a crown ether.

6. The modified polypeptide of claim 2, wherein said functional group is a naturally occurring molecule.

7. The modified polypeptide of claim 6, wherein said functional group is an enzyme inhibitor, a hapten, a nucleotide, an amino acid, a lipid, a toxin, a saccharide, a chelator or a cyclodextrin.

8. The modified polypeptide of claim 1, wherein said sensing moiety is a polymer.

9. The modified polypeptide of claim 8, wherein said polymer is polyethylene glycol (PEG).

10. The modified polypeptide of claim 9, wherein said polymer is polyethylene glycol (PEG)-biotin.

11. The modified polypeptide of claim 8, wherein said polymer is an analyte-binding polymer.

12. The modified polypeptide of claim 11, wherein said polymer is an oligonucleotide, an oligosaccharide or a peptide.

13. The modified polypeptide of claim 1, wherein said sensing moiety binds to a metal, metal ion, a toxin, an enzyme, a nucleotide, an oligonucleotide, an amino acid, a peptide, a saccharide, a hapten, a lipid or an antibody or antigen-binding fragment thereof.

14. The modified polypeptide of claim 1, wherein said sensing moiety responds to a change in the type or amount of a biological or chemical constituent in the environment of said oligomeric pore assembly.

15. The modified polypeptide of claim 1, wherein said sensing moiety responds to a change in the physical environment of said oligomeric pore assembly.

16. The modified polypeptide of claim 15, wherein said sensing moiety responds to a change in pH, light, voltage or temperature.

17. The modified polypeptide of claim 1, wherein said polypeptide is covalently linked to at least a first and at least a second sensing moiety.

18. The modified polypeptide of claim 17, wherein said at least a first sensing moiety is distinct from said at least a second sensing moiety.

19. The modified polypeptide of claim 17, wherein said at least a first sensing moiety is the same as said at least a second sensing moiety.

20. The modified polypeptide of claim 1, wherein said polypeptide is a staphylococcal hemolysin polypeptide, a porin, a complement pore polypeptide, a hemolysin C polypeptide or a streptolysin O polypeptide.

21. The modified polypeptide of claim 20, wherein said polypeptide is a staphylococcal alpha hemolysin polypeptide.

22. The modified polypeptide of claim 21, wherein said polypeptide is a mutant staphylococcal alpha hemolysin polypeptide comprising at least a first heterologous amino acid.

23. The modified polypeptide of claim 22, wherein said mutant staphylococcal alpha hemolysin polypeptide comprises a cysteine residue in place of serine at position 106 of the wild-type staphylococcal alpha hemolysin polypeptide or a cysteine residue in place of lysine at position 8 of the wild-type staphylococcal alpha hemolysin polypeptide.

24-43. -43. (canceled)