Methods for generating pH/ionic concentration gradient near electrode surfaces for modulating biomolecular interactions

• US 9,874,538 B2
• Filed: 07/06/2015
• Issued: 01/23/2018
• Est. Priority Date: 07/06/2012
• Status: Active Grant

• Current Assignee: Robert Bosch GmbH
• Original Assignee: Robert Bosch GmbH
• Inventors: Johnson, Christopher, Kavusi, Sam, Gangadharan, Rajan, Verma, Piyush, Fomina, Nadezda, Ahmad, Habib, Lau, I, Aldrich
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US14/792,569
• Publication Number:
  US 20160003766A1
• Time in Patent Office: 932 Days
• CPC Class Codes: G01N 27/3271 Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001
• CPC Class Codes: G01N 27/3276 being a hybridisation with immobilised receptors using a FET type sensor G01N27/4145; concerning the hybridisation C12Q1/68
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding

Abstract

Device and methods for use in a biosensor comprising a multisite array of test sites, the device and methods being useful for modulating the binding interactions between a (biomolecular) probe or detection agent and an analyte of interest by modulating the pH or ionic gradient near the electrodes in such biosensor. An electrochemically active agent that is suitable for use in biological buffers for changing the pH of the biological buffers. Method for changing the pH of biological buffers using the electrochemically active agents. The methods of modulating the binding interactions provided in a biosensor, analytic methods for more accurately controlling and measuring the pH or ionic gradient near the electrodes in such biosensor, and analytic methods for more accurately measuring an analyte of interest in a biological sample.

Claims

1. A method comprising:

a. adding an electrochemically active agent which is a quinone derivative onto a support of a biosensor; and

b. adding an analyte that, in dependence upon a change of a redox state of the quinone derivative, interacts with a probe on the support, wherein an electrode of the support is configured to cause the change in the redox state of the quinone derivative.

2. The method of claim 1, wherein the change of the redox state of the quinone derivative allows the interaction between the analyte and the probe to occur.

3. The method of claim 1, wherein the change of the redox state of the quinone derivative increases a binding affinity between the analyte and the probe.

4. The method of claim 1, wherein the change of the redox state of the quinone derivative decreases a binding affinity between the analyte and the probe.

5. A method comprising:

a. providing a biosensor comprising a support in an aqueous solution, wherein;

i. the support comprising one or more electrodes, and a biomolecule interface layer having one or more immobilized probes thereon; and

ii. the aqueous solution comprising a quinone derivative;

b. adding a biomolecule analyte to the aqueous solution;

c. reacting the quinone derivative using the one or more electrodes to produce an amount of H⁺ ions and/or an amount of OH⁻

ions, wherein the pH of the aqueous solution close to the one or more electrodes is controlled by the amount of H⁺ ions and/or the amount of OH⁻

ions produced; and

d. collecting signals from the biosensor.

6. The method of claim 5, wherein the one or more immobilized probes comprises a pH sensitive probe.

7. The method of claim 6, wherein the pH sensitive probe is a fluorescent protein and optionally a green fluorescent protein.

8. The method of claim 6, wherein a first area of the biomolecule interface covers at least one area of the support not covered by the one or more electrode and a second area of the biomolecule interface covers at least one area of the one or more electrodes.

9. The method of claim 8, wherein the pH sensitive probe is immobilized on the first area and second area of the biomolecule interface.

10. The method of claim 9, further comprising determining the pH of the solution near the one or more electrode using a fluorescence intensity of an immobilized pH sensitive fluorescent protein immobilized on the second area of the biomolecule interface layer.

11. The method of claim 9, further comprising normalizing the fluorescence intensity of the immobilized pH sensitive fluorescent protein immobilized on the second area of the biomolecule interface layer with respect to the fluorescence intensity of the immobilized pH sensitive fluorescent protein immobilized on the first area of the biomolecule interface layer, and the pH is determined using the normalized fluorescence intensity.

12. The method of claim 5, wherein the one or more immobilized probes comprises an immobilized enzyme and the biomolecule analyte is an enzyme substrate.

13. The method of claim 5, wherein the one or more immobilized probes comprises an immobilized enzyme substrate and the biomolecule analyte is an enzyme.

14. The method of claim 5, further comprising measuring the pH using a sense electrode wherein the one or more electrodes comprises the sense electrode.

15. The method of claim 5, wherein the biosensor comprises a multisite array of test sites with each test site having the support in the solution and wherein one or more test conditions for each test site can be independently varied.

16. The method of claim 15, wherein the pH of the solution close to the one or more electrodes in each test site is independently controlled.

17. The method of claim 15, wherein collecting signals from the biosensor include collecting varied signals from the multisite array of test sites to obtain a collection of varied signals.

18. A biosensor system comprising:

a. a support that includes an electrode;

b. a material on the support;

c. an electrochemically active agent in an aqueous solution, wherein the electrode is configured to control a change of a redox state of the electrochemically active agent, and the material provides an indication of the change of the redox state.

19. A biosensor system comprising:

a. a support that includes an electrode;

b. a probe on the support;

c. an electrochemically active agent which is a quinone derivative; and

d. an analyte that interacts with the probe on the support in dependence upon a change of a redox state of the quinone derivative, wherein the electrode is configured to control the change of the redox state of the quinone derivative.

20. The biosensor of claim 19, wherein the change of the redox state of the quinone derivative allows the interaction between the analyte and the probe to occur.

21. The biosensor of claim 19, wherein the change of the redox state of the quinone derivative increases a binding affinity between the analyte and the probe.

22. The biosensor of claim 19, wherein the change of the redox state of the quinone derivative decreases a binding affinity between the analyte and the probe.

23. The biosensor of claim 19, wherein:

the support includes a biomolecule interface layer;

a first area of the biomolecule interface layer covers at least one area of the support not covered by the electrode; and

a second area of the biomolecule interface layer covers at least one area of the electrode.

24. A biosensor comprising a support in a solution, wherein:

a. the solution comprises a quinone derivative;

b. the support comprises one or more electrodes and a biomolecule interface layer having one or more immobilized probes thereon; and

c. the biosensor is configured to modify the pH of the solution near the one or more electrodes by electrochemically oxidizing or reducing the quinone derivative to produce H⁺ ions or OH⁻

ions.

25. The biosensor of claim 24, wherein the solution is an aqueous solution.

26. The biosensor of claim 24, wherein the one or more immobilized probes comprises a pH sensitive probe.

27. The biosensor of claim 26, wherein the pH sensitive probe is a fluorescent protein and optionally a green fluorescent protein.

28. The biosensor of claim 26, wherein a first area of the biomolecule interface covers at least one area of the support not covered by the one or more electrode and a second area of the biomolecule interface covers at least one area of the one or more electrodes.

29. The biosensor of claim 28, wherein the pH sensitive probe is immobilized on the first area and second area of the biomolecule interface.

30. The biosensor of claim 29, wherein the biosensor is configured to normalize the fluorescence intensity of the immobilized pH sensitive fluorescent protein immobilized on the second area of the biomolecule interface layer with respect to the fluorescence intensity of the immobilized pH sensitive fluorescent protein immobilized on the first area of the biomolecule interface layer, and the biosensor is configured to determine the pH using the normalized fluorescence intensity.

31. The biosensor of claim 29, wherein the biosensor is configured to determine the pH of the solution near the one or more electrode using a fluorescence intensity of an immobilized pH sensitive fluorescent protein immobilized on the second area of the biomolecule interface layer.

32. The biosensor of claim 24, wherein the one or more immobilized probes comprises an immobilized enzyme and the biomolecule analyte is an enzyme substrate.

33. The biosensor of claim 24, wherein the one or more immobilized probes comprises an immobilized enzyme substrate and the biomolecule analyte is an enzyme.

34. The biosensor of claim 24, wherein the one or more electrodes comprises a sense electrode and the biosensor is configured to measure the pH using the sense electrode.

35. The biosensor of claim 24, wherein the biosensor comprises a multisite array of test sites with each test site having the support in the solution and one or more test conditions for each test site can be independently varied.

36. The biosensor of claim 35, wherein the pH of the solution close to the one or more electrodes in each test site is independently controlled.

37. The biosensor of claim 35, wherein the biosensor is configured to obtain a collection of varied signals including varied signals from the multisite array of test sites.