Biosensor for small molecule analytes

• US 20060292581A1
• Filed: 08/09/2005
• Published: 12/28/2006
• Est. Priority Date: 08/20/2001
• Status: Abandoned Application

• Current Assignee: Lance G. Laing
• Original Assignee: Lance G. Laing
• Inventors: Laing, Lance G.
• Application Number:
  US11/200,892
• Publication Number:
  US 20060292581A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: C12Q 2565/628 being a surface plasmon resonance spectrometer

Abstract

A biosensor device for detecting small molecules analytes is provided. The device employs a first class of molecules, e.g., protein that binds to both the analyte and a second class of molecules, e.g., nucleic acid. The binding of the protein to the analyte and nucleic acid can be mutually exclusive, and the presence of analyte in a sample results in a detectable displacement of protein from nucleic acid. Alternatively, binding of the protein to the nucleic acid can depend on the presence of analyte in the sample. In a specific embodiment, either the protein or nucleic acid is immobilized on a solid phase support. An arsenic detection system is exemplified. An ArsR binding sequence from the E. coli ars operon is immobilized on a gold-plated surface. ArsR protein binds to the DNA in the absence of arsenic, and is released in the presence of sodium arsenate or phenylarsine oxide. Protein release results in a change in surface plasmon resonance, and the magnitude or kinetics of the change indicate the concentration of arsenic.

Claims

1. A composition for detecting the presence of an analyte in a sample comprising (i) a purified protein that specifically binds the analyte (ii) a purified nucleic acid containing a specific binding sequence which is bound specifically by the protein;

and (iii) a detection system that indicates a change in binding of the protein to the nucleic acid in the presence of the analyte, which detection system is selected from the group consisting of a surface plasmon resonance system, electrochemical transducer, optical transducer, colorimetric-based system, fluorescent-based system, sandwich assay and electrochemical system.

2. The composition of claim 1, wherein protein binding to the nucleic acid and the analyte is mutually exclusive, such that the protein bound to the nucleic acid is released from the nucleic acid upon binding the analyte.

3. The composition of claim 2, wherein the protein bound to the nucleic acid undergoes an allosteric change upon binding the analyte.

4. The composition of claim 1, wherein the nucleic acid is a DNA.

5. The composition of claim 1, wherein the analyte is a metal compound selected from the group consisting of Ag, As, Ba, Cd, Cr, Hg, Pb, and Se.

6. The composition of claim 5, wherein the metal compound is As.

7. The composition of claim 1, wherein the protein is a bacterial DNA-binding regulatory protein encoded by a metal-response operon and the nucleic acid is a DNA containing a specific binding sequence from the metal-response operon promoter that is specifically bound by the protein.

8. The composition of claim 7, wherein the metal compound is selected from the group consisting of Ag, As, Cd, Cr, Hg, and Pb.

9. The composition of claim 7, wherein the metal compound is As, the protein is ArsR, and the promoter is an ars operon promoter.

10. A biosensor device for detecting the presence of an analyte in a sample comprising (i) a protein that specifically binds the analyte;

(ii) a purified nucleic acid containing a specific binding sequence which is bound specifically by the protein; and

(iii) a detection system that indicates a change in binding of the protein to the nucleic acid in the presence of the analyte, which detection system is selected from the group consisting of a surface plasmon resonance system, electrochemical transducer, optical transducer, colorimetric-based system, fluorescent-based system, sandwich assay and electrochemical system;

wherein either the protein or the nucleic acid is bound to a solid phase support.

11. The device of claim 10, wherein protein binding to the nucleic acid and the analyte is mutually exclusive, such the protein bound to the nucleic acid is release from the nucleic acid upon binding the analyte.

12. The device of claim 10, wherein the detection system is a surface plasmon resonance system.

13. The device of claim 10, wherein the protein is a bacterial DNA-binding regulatory protein encoded by a metal-response operon and the nucleic acid is a DNA containing a specific binding sequence from the metal-response operon promoter that is specifically bound by the metal compound.

14. The device of claim 13, wherein the analyte is a metal compound selected from the group consisting of Ag, As, Cd, Cr, Hg, and Pb.

15. The device of claim 14, wherein the metal compound is As, the protein is ArsR, and the promoter is an ars operon promoter.

16. The device of claim 15, wherein the ars operon is a an E. coli ars operon.

17. The device of claim 10, wherein the solid support is in contact with a flow cell that permits flow of liquid over the solid support.

18. The device of claim 17, wherein the solid support comprises a metal film that forms an interface with liquid, wherein the detection system is a surface plasmon resonance system.

19. The device of claim 18, wherein the solid support is a silicon chip of less than 1 cm square area.

20. The device of claim 18, which device maintains temperature stability when sample is applied.

21. The device of claim 18, further comprising a microprocessor capable of processing changes in plasmon resonance at the surface over time.

22. A method of detecting the presence of an analyte in a sample, which method comprises:

a. contacting the sample with a protein that specifically binds the analyte, which protein is bound to a purified nucleic acid containing a specific binding sequence for the protein, wherein binding of the protein to the analyte is mutually exclusive of binding to the nucleic acid; and

b. detecting release of the protein from the nucleic acid by use of a detection system selected from the group consisting of a surface plasmon resonance system, electrochemical transducer, optical transducer, colorimetric-based system, fluorescent-based system, sandwich assay and electrochemical system.

23. The method according to claim 22, wherein the protein undergoes an allosteric change upon binding the analyte.

24. The method according to claim 22, wherein the nucleic acid is a DNA.

25. The method of claim 24, wherein the analyte is a metal compound selected from the group consisting of Ag, As, Ba, Cd, Cr, Hg, Pb, and Se.

26. The method according to claim 25, wherein the metal compound is As.

27. The method according to claim 22, wherein the protein is a bacterial DNA-binding regulatory protein encoded by a metal-response operon and the nucleic acid is a DNA containing a specific binding sequence from the metal-response operon promoter that is specifically bound by the protein.

28. The method according to claim 27, wherein the analyte is a metal compound selected from the group consisting of Ag, As, Cd, Cr, Hg, and Pb.

29. The method according to claim 22, wherein the protein comprises a sequence of a bacterial DNA-binding regulatory protein encoded by a metal-response operon sufficient to bind DNA and the metal.

30. The method according to claim 22, wherein the nucleic acid is a DNA containing a sequence that is modified to differ by at least one nucleotide from a specific binding sequence from a metal-response operon promoter that is specifically bound by the protein, and which DNA is specifically bound by the protein with different binding constants than the unmodified sequence.

31. The method according to claim 29, wherein the nucleic acid is a DNA containing a sequence that is modified to differ by at least one nucleotide from a specific binding sequence from a metal-response operon promoter that is specifically bound by the protein, and which DNA is specifically bound by the protein with different binding constants than the unmodified sequence.

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. (canceled)

37. (canceled)