Stochastic sensing through covalent interactions

• US 8,404,449 B2
• Filed: 04/24/2009
• Issued: 03/26/2013
• Est. Priority Date: 05/10/2002
• Status: Active Grant

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Bayley, Hagan, Shin, Seong-Ho, Luchian, Tudor, Cheley, Stephen
• Primary Examiner(s): LAM, ANN Y
• Application Number:
  US12/429,897
• Publication Number:
  US 20090283422A1
• Time in Patent Office: 1,432 Days
• Field of Search: None
• 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 method for sensing an individual molecule of at least one reactive analyte in a solution comprising:

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

providing a detection system operable to detect a 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 within the lumen of the pore and thereby altering the current between the cis and trans chambers to the cis or trans chamber; and

detecting the current between the cis and trans chambers, wherein a change in the current between the cis and trans chambers indicates whether the analyte is covalently bound to the protein pore; and

using the detected current, stochastically sensing the at least one analyte.

2. The method of claim 1, wherein the pore is an α

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

3. The method of claim 1, wherein the reactive analyte is selected from the group consisting of:

chemical weapons, environmental toxins and pharmaceuticals.

4. The method of claim 1, wherein the reactive analyte comprises an arsenical.

5. The method of claim 1, 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.

6. The method of claim 5, wherein the reactive analyte and chemical reagent are provided to separate chambers.

7. The method of claim 1, 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.

8. The method of claim 7, further comprising analyzing the detected current between the cis and trans chambers for the distinct current signals.

9. The method of claim 1, further comprising determining the identity and concentration of at least one reactive analyte based upon the detected current between the cis and trans chambers.

10. The method of claim 1, wherein the reactive analyte irreversibly covalently bonds to the protein pore, further comprising providing a chemical capable of reacting with the analyte to produce a second signal.

11. A method for sensing an individual molecule of at least one reactive analyte in a solution comprising:

introducing an ionic solution containing at least one reactive analyte capable of covalently bonding to an engineered protein pore within the lumen of the pore into a sensing device comprising a trans chamber and a cis chamber separated by a divider, wherein the divider comprises the engineered protein pore operably disposed therein;

detecting a change in current between the cis and trans chambers, wherein the change in the current between the cis and trans chambers indicates whether the analyte is covalently bound to the protein pore;

using the change in detected current to sense an individual molecule of the reactive analyte.

12. The method of claim 11, wherein the pore is an α

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

13. The method of claim 11, wherein the reactive analyte is selected from the group consisting of:

chemical weapons, environmental toxins and pharmaceuticals.

14. The method of claim 11, wherein the reactive analyte comprises an arsenical.

15. The method of claim 11, wherein the reactive analyte irreversibly covalently bonds to the protein pore, further comprising breaking the covalent bond using a chemical reagent.

16. The method of claim 15, further comprising placing the reactive analyte in one of the cis or trans chambers and placing the chemical reagent in the other chamber.

17. The method of claim 11, 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.

18. The method of claim 17, further comprising analyzing the change in detected current between the cis and trans chambers for the distinct current signals.

19. The method of claim 11, wherein using the change in detected current to sense an individual molecule of the reactive analyte comprises determining the identity and concentration of at least one reactive analyte.

20. The method of claim 11, wherein the reactive analyte irreversibly covalently bonds to the protein pore, further comprising introducing a chemical capable of reacting with the analyte to produce a second signal.