Methods for generating pH/ionic concentration gradient near electrode surfaces for modulating biomolecular interactions, and bubble detection using electrodes

• US 9,766,197 B2
• Filed: 07/06/2015
• Issued: 09/19/2017
• Est. Priority Date: 02/13/2014
• Status: Active Grant

• Current Assignee: Robert Bosch GmbH
• Original Assignee: Robert Bosch GmbH
• Inventors: Johnson, Christopher, Kavusi, Sam, Gangadharan, Rajan, Verma, Piyush, Lau, Aldrich, Funk, Tobias
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US14/792,530
• Publication Number:
  US 20160003763A1
• Time in Patent Office: 806 Days
• CPC Class Codes: G01N 27/226 Construction of measuring vessels; Electrodes therefor
• CPC Class Codes: G01N 27/228 Circuits therefor measuring capacitance per se G01R27/26
• 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 27/3272 Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels optical biosensors G01N33/52
• CPC Class Codes: G01N 27/3274 Corrective measures, e.g. error detection, compensation for temperature or hematocrit, calibration coding of calibration information G01N33/48771
• CPC Class Codes: G01N 27/3276 being a hybridisation with immobilised receptors using a FET type sensor G01N27/4145; concerning the hybridisation C12Q1/68

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 from a biological 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. providing a biosensor comprising;

i. a control unit and a support in a solution,

wherein the solution comprises an electrochemically active agent, and

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

b. modifying the pH of the solution near at least one of the two or more electrodes by electrochemically oxidizing or reducing the electrochemically active agent to produce H⁺ ions or Off ions;

c. measuring capacitance values associated with at least two of the two or more electrodes; and

d. determining based on the measured capacitance values whether a bubble is present in the solution.

2. The method of claim 1, wherein the solution is an aqueous solution.

3. The method of claim 1, wherein the one or more immobilized probes comprises a pH sensitive probe.

4. The method of claim 3, 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.

5. The method of claim 4, wherein the pH sensitive probe is immobilized on the first area and second area of the biomolecule interface.

6. The method of claim 5, 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.

7. The method of claim 5, 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.

8. The method of claim 3, wherein the pH sensitive probe is a fluorescent protein and optionally a green fluorescent protein.

9. The method of claim 1, wherein the one or more immobilized probes comprises an immobilized enzyme and the biomolecule analyte is an enzyme substrate.

10. The method of claim 1, wherein the one or more immobilized probes comprises an immobilized enzyme substrate and the biomolecule analyte is an enzyme.

11. The method of claim 1, further comprising measuring the pH using a sense electrode wherein the one or more electrodes comprises the sense electrode.

12. The method of claim 1, 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 condition for each test site can be independently varied.

13. The method of claim 12, wherein the pH of the solution close to the one or more electrodes in each test site is independently controlled.

14. The method of claim 12, further comprising collecting signals from the biosensor to obtain a collection of varied signals, wherein the collection of varied signals includes varied signals from the multisite array of test sites.

15. The method of claim 12, wherein the control unit is configured to independently detect a presence of a bubble in each test site and if the presence of a bubble is detected store the location of the test site in the multisite array having the bubble.

16. A biosensor comprising:

a. a support in a solution,

i. the solution comprising an electrochemically active agent,

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

b. a control unit,

c. wherein the biosensor is configured to modify the pH of the solution near at least one of the two or more electrodes by electrochemically oxidizing or reducing the electrochemically active agent to produce H⁺ ions or OH⁻

ions, and the control unit is configured to measure a capacitance values associated with at least two of the two or more electrodes and determine based on the measured capacitance values whether a bubble is present in the solution.

17. The biosensor of claim 16, wherein the solution is an aqueous solution.

18. The biosensor of claim 16, wherein the one or more immobilized probes comprises a pH sensitive probe.

19. The biosensor of claim 18, wherein the pH sensitive probe is a fluorescent protein and optionally a green fluorescent protein.

20. The biosensor of claim 18, 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.

21. The biosensor of claim 20, wherein the pH sensitive probe is immobilized on the first area and second area of the biomolecule interface.

22. The biosensor of claim 21, wherein the biosensor is configured to determine the pH of the solution near the one or more electrode using a fluorescence intensity of the immobilized pH sensitive fluorescent protein immobilized on the second area of the biomolecule interface layer.

23. The biosensor of claim 21, 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.

24. The biosensor of claim 16, wherein the one or more immobilized probes comprises an immobilized enzyme and the biomolecule analyte is an enzyme substrate.

25. The biosensor of claim 16, wherein the one or more immobilized probes comprises an immobilized enzyme substrate and the biomolecule analyte is an enzyme.

26. The biosensor of claim 16, wherein the one or more electrodes comprises a sense electrode and the biosensor is configured to measure the pH using the sense electrode.

27. The biosensor of claim 16, 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 condition for each test site can be independently varied.

28. The biosensor of claim 27, wherein the pH of the solution close to the one or more electrodes in each test site is independently controlled.

29. The biosensor of claim 27, wherein the control unit is configured to independently detect a presence of a bubble in each test site and if the presence of a bubble is detected store the location of the test site in the multisite array having the bubble.

30. The biosensor of claim 27, wherein the biosensor is configured to obtain a collection of varied signals including varied signals from the multisite array of test sites.