Biosensor compositions and methods of use

• US 6,916,665 B2
• Filed: 02/12/2001
• Issued: 07/12/2005
• Est. Priority Date: 02/11/2000
• Status: Expired due to Term

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Bayley, Hagan P., Movileanu, Liviu, Howorka, Stefan G.
• Primary Examiner(s): Ponnaluri, Padmashri
• Assistant Examiner(s): TRAN, MY CHAU T
• Application Number:
  US09/781,697
• Publication Number:
  US 20020094526A1
• Time in Patent Office: 1,611 Days
• Field of Search: 435/4, 435/6, 435/7.2, 435/287.1, 435/287.2, 436/86, 436/518, 436/528, 436/531, 422/50, 422/68.1, 422/82.01, 422/99, 530/350
• US Class Current: 436/149
• 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 method of detecting the presence of an analyte in a sample, the method comprising:

contacting said sample with a pore assembly comprising one or more pore-subunit polypeptides sufficient to form a pore, wherein the pore comprises at least a first channel, and at least one of said pore-subunit polypeptides is a modified pore-subunit polypeptide comprising a pore-subunit polypeptide covalently linked to an exogenous sensing moiety capable of preferentially binding with a specific analyte; and

detecting an electrical current through at least a first channel, wherein a modulation in current compared to a current measurement in a control sample lacking said analyte indicates the presence of said analyte in said sample.

2. The method of claim 1, wherein said electrical current is detected through a single channel.

3. The method of claim 1, wherein said electrical current is detected through at least two channels.

4. The method of claim 1, wherein said analyte is known.

5. The method of claim 1, wherein said analyte is unknown.

6. The method of claim 1, wherein said analyte is an oligonucleotide.

7. The method of claim 1, wherein the amount of said analyte in said sample is quantitated.

8. The method of claim 1, wherein the exogenous sensing moiety is a polymer.

9. The method of claim 1, wherein the exogenous sensing moiety is an oligonucleotide or a polynucleotide.

10. The method of claim 1, wherein the exogenous sensing moiety is a single stranded DNA molecule.

11. The method of claim 1, wherein the modified pore-subunit polypeptide is a pore-subunit polypeptide covalently linked to an oligonucleotide.

12. The method of claim 11 wherein the modified pore-subunit polypeptide is a staphylococcal alpha hemolysin pore-subunit polypeptide covalently linked to an oligonucleotide.

13. The method of claim 1, wherein the exogenous sensing moiety is an oligonucleotide and wherein the analyte comprises a polynucleic acid comprising a base sequence that is complementary to the exogenous sensing moiety.

14. A method of detecting the presence of an analyte in a sample, wherein the analyte comprises a polynucleic acid comprising a specific base sequence, the method comprising:

contacting said sample with a pore assembly comprising one or more pore-subunit polypeptides sufficient to form a pore, wherein the pore comprises at least a first channel, and at least one of said pore-subunit polypeptides is a modified pore-subunit polypeptide comprising a pore-subunit polypeptide covalently linked to an exogenous sensing moiety that is an oligonucleotide, wherein the oligonucleotide comprises a base sequence that is complementary to said specific base sequence of said analyte; and

detecting an electrical current through at least a first channel, wherein a modulation in current compared to a current measurement in a control sample lacking said analyte indicates the presence of said analyte in said sample.