Stochastic sensing through covalent interactions

• US 20030215881A1
• Filed: 05/09/2003
• Published: 11/20/2003
• Est. Priority Date: 05/10/2002
• Status: Abandoned Application

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Cheley, Stephen, Bayley, Hagan, Luchian, Tudor, Shin, Seong-Ho
• Application Number:
  US10/434,897
• Publication Number:
  US 20030215881A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

A system and method for stochastic sensing in which the analyte covalently bonds to the sensor element or an adaptor element. If such bonding is irreversible, the bond may be broken by a chemical reagent. The sensor element may be a protein, such as the engineered P_SH type or αHL protein pore. The analyte may be any reactive analyte, including chemical weapons, environmental toxins and pharmaceuticals. The analyte covalently bonds to the sensor element to produce a detectable signal. Possible signals include change in electrical current, change in force, and change in fluorescence. Detection of the signal allows identification of the analyte and determination of its concentration in a sample solution. Multiple analytes present in the same solution may be detected.

Claims

1. A system for sensing at least one reactive analyte in a solution comprising:

a sensing device separated into a trans chamber and a cis chamber by a divider;

a protein pore operably disposed in the divider;

a detection system operable to detect current between the cis and trans chambers; and

an ionic solution containing at least one reactive analyte capable of covalently bonding to the protein pore;

wherein bonding of the reactive analyte to the protein pore produces a change in current between the cis and trans chambers detectable by the current detection system.

2. The system of claim 1, wherein the protein pore is an engineered protein pore.

3. The system of claim 1, wherein the protein pore is an α

HL pore.

4. The system of claim 3, wherein the α

HL pore comprises at least one monomer having a Cys residue at amino acid 117.

5. The system of claim 1, wherein the ionic solution is a pH buffered KCl solution.

6. The system of claim 1, wherein the reactive analyte is an environmental toxin.

7. The system of claim 1, wherein the reactive analyte is a chemical weapon.

8. The system of claim 1, wherein the reactive analyte is a pharmaceutical.

9. The system of claim 1, wherein the reactive analyte comprises an arsenical.

10. The system of claim 1, wherein the reactive analyte covalently bonds to the protein pore within the lumen of the pore.

11. The system of claim 1, wherein the reactive analyte irreversibly covalently bonds to the protein pore, further comprising an ionic solution containing a chemical reagent capable of breaking the covalent bond.

12. The system of claim 11, wherein the reactive analyte and chemical reagent are substantially disposed in separate chambers.

13. The system of claim 1, wherein the ionic solution contains a plurality of species of chemically distinct reactive analytes capable of covalently bonding with the protein pore.

14. The system of claim 13, wherein bonding of at least one species of the reactive analytes to the protein pore produces a first change in current between the cis and trans chambers and the bonding of at least a second species of the reactive analytes to the protein pore produces a second change in current between the cis and trans chambers, wherein the first and second changes in current are distinctly detectable by the detection system.

15. A system for sensing at least one reactive analyte in a sample comprising:

a sensor element; and

a sample containing at least a first reactive analyte, wherein the reactive analyte covalently bonds to the sensor element.

16. The system of claim 15, wherein sensing comprises stochastic sensing.

17. The system of claim 15, wherein the sensor element produces a detectable signal when covalently bound to the reactive analyte.

18. The system of claim 15, wherein the sensor element is a protein.

19. The system of claim 18, wherein the sensor element is a protein pore.

20. The system of claim 15, wherein the analyte directly covalently bonds to the sensor element.

21. The system of claim 15, wherein the analyte covalently bonds to an adaptor molecule that bonds to the sensor element.

22. The system of claim 15, wherein the reactive analyte is selected from the group consisting of:

chemical weapons, environmental toxins and pharmaceuticals.

23. The system of claim 15 wherein the reactive analyte irreversibly covalently bonds to the sensor element, further comprising a chemical reagent capable of breaking the covalent bond.

24. The system of claim 15, further comprising at least a second reactive analyte, wherein the second reactive analyte covalently bonds to the sensor element.

25. The system of claim 15, further comprising a detection system operable to detect a signal produced by covalent bonding of the reactive analyte to the sensor element.

26. The system of claim 25, wherein the signal is selected from the group consisting of:

a change in electrical current, a change in force, and a change in fluorescence.

27. The system of claim 25, wherein the signal comprises a change in the magnitude of an electrical current.

28. The system of claim 25, further comprising:

at least a second reactive analyte, wherein the second reactive analyte covalently bonds to the sensor element; and

a detection system operable to detect a signal produced by covalent bonding of of a reactive analyte to the sensor element,

wherein the detection system is capable of distinctly detecting the signals produced by covalent bonding of at least the first and second reactive analytes to the sensor element.

29. The system of claim 15, further comprising a plurality of sensor elements.

30. The system of claim 25, wherein the first and second reactive analytes covalently bond to two different binding sites on the sensor element.

31. A method for sensing at least one analyte in a solution comprising:

providing a sensing device separated into a trans chamber and a cis chamber by a divider, wherein a protein pore is operably disposed in the divider;

providing a detection system operable to detect current between the cis and trans chambers;

providing an ionic solution containing at least one reactive analyte capable of covalently bonding to the protein pore to the cis or trans chamber; and

detecting the current between the cis and trans chambers.

32. The method of claim 31, wherein the pore is an engineered protein pore.

33. The method of claim 31, wherein the pore is an α

HL pore comprising at least one monomer having a Cys residue at amino acid 117.

34. The method of claim 31, wherein the reactive analyte is selected from the group consisting of:

chemical weapons, environmental toxins and pharmaceuticals.

35. The method of claim 31, wherein the reactive analyte comprises an arsenical.

36. The method of claim 31, wherein the reactive analyte covalently bonds to the protein pore within the lumen of the pore.

37. The method of claim 31, wherein the reactive analyte irreversibly covalently bonds to the protein pore, further comprising providing to the cis or trans chamber an ionic solution containing a chemical reagent capable of breaking the covalent bond.

38. The method of claim 37, wherein the reactive analyte and chemical reagent are provided to separate chambers.

39. The method of claim 31, wherein the ionic solution comprises a plurality of species of chemically distinct reactive analytes capable of covalently bonding with the protein pore to produce distinct current signals.

40. The method of claim 39, further comprising analyzing the detected conductance between the cis and trans chambers for the distinct current signals.

41. The method of claim 31, further comprising determining the identity and concentration of at least one reactive analyte based upon the current between the cis and trans chambers.

42. The method of claim 31, wherein the reactive analyte irreversibly covalently bonds to the protein pore, further comprising a chemical capable of reacting with the analyte to produce a second signal.

43. A method for sensing at least one reactive analyte in a sample comprising:

providing a sensor element;

providing a sample containing at least a first reactive analyte capable of covalently bonding to the sensor element;

allowing the reactive analyte to covalently bond to the sensor element to produce a first signal; and

detecting the signal.

44. The method of claim 43, wherein sensing comprises stochastic sensing.

45. The method of claim 43, wherein the sensor element is a protein.

46. The method of claim 43, wherein the sensor element is an engineered protein.

47. The method of claim 43, wherein the reactive analyte is selected from the group consisting of:

chemical weapons, environmental toxins, and pharmaceuticals.

48. The method of claim 43, wherein the reactive analyte irreversibly covalently bonds to the sensor element, further comprising allowing a chemical reagent to break the covalent bond.

49. The method of claim 43, further comprising:

allowing at least a second reactive analyte to covalently bond to the sensor element to produce a second signal, wherein the second signal is distinct from the first signal; and

detecting the second signal.

50. The method of claim 43, further comprising providing a plurality of sensor elements.

51. The method of claim 43, further comprising comparing the detected signal to a known set of signals to determine the identity of the reactive analyte.

52. The method of claim 43, further comprising:

determining the frequency of detection of the signal; and

calculating the concentration of the reactive analyte based upon the frequency of detection of the signal.

53. The method of claim 43, wherein the signal is selected from the group consisting of:

change in electrical current, change in force, and change in fluorescence.