Strip-based electrochemical sensors for quantitative analysis of analytes

• US 10,364,452 B2
• Filed: 06/01/2015
• Issued: 07/30/2019
• Est. Priority Date: 05/30/2014
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Wang, Joseph, Litvan, Irene, Windmiller, Joshua Ray
• Primary Examiner(s): Noguerola, Alexander S
• Application Number:
  US15/312,733
• Publication Number:
  US 20170226557A1
• Time in Patent Office: 1,520 Days
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• 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 27/3273 Devices therefor, e.g. test element readers, circuitry details not specific to biochemical electrodes G01N33/4875
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

Methods, systems, and devices are disclosed for providing test strip electrochemical sensors for rapid quantitative analysis of analytes in physiological fluids. In one aspect, an electrochemical sensor includes a substrate; an electrode contingent including a first electrode having a coating of carbon nanotubes (CNTs) and a second electrode on the substrate; and an entrapment layer formed on the first electrode to attach an enzymatic substance capable of causing a redox reaction in the presence of a target analyte of a fluid sample which produces a redox-active product, in which the entrapment layer is structured to include a conductive polymer film that is reversibly dopable to conduct charge carriers across the entrapment layer and at the first electrode. For example, the electrochemical sensor can be provided on a disposable test strip to quantify L-DOPA levels in whole blood, plasma, or serum samples.

Claims

1. An electrochemical sensor, comprising:

a substrate formed of an insulative material;

an electrode contingent including a first electrode and a second electrode on the substrate, wherein the first electrode includes a coating of carbon nanotubes (CNTs); and

an entrapment layer formed on the first electrode to attach an enzymatic substance capable of causing a redox reaction in the presence of a target analyte of a fluid sample which produces a redox-active product, wherein the entrapment layer is structured to include a conductive polymer film that is reversibly dopable to conduct charge carriers across the entrapment layer and at the first electrode,

wherein the conductive polymer film includes poly(thionine).

2. The electrochemical sensor of claim 1, wherein the electrochemical sensor is operable for electrochemical analysis of a fluid sample when the fluid sample is in contact with the electrode contingent and the electrochemical sensor is electrically coupled to an electrical circuit to supply an electrical waveform to the electrode contingent to transduce chemical information associated with the target analyte that is present in the fluid sample to an electrical signal.

3. The electrochemical sensor of claim 2, wherein the electrical signal indicates a concentration level of the target analyte in the fluid sample.

4. The electrochemical sensor of claim 3, wherein the magnitude of the electrical signal with respect to time changes in proportion to the concentration of the target analyte.

5. The electrochemical sensor of claim 1, wherein the electrochemical sensor is formed on a disposable test strip.

6. The electrochemical sensor of claim 5, further comprising electrode interface contacts on the substrate that are electrically coupled to the electrodes via electrically conductive conduits, wherein the electrode interface contacts are operable to provide electrical signals detected by the electrode contingent when the disposable electrochemical test strip sensor is electrically interfaced with an electrochemical analyzer device.

7. The electrochemical sensor of claim 6, further comprising an electrically insulative cover layer at least partially formed over the electrically conductive conduits.

8. The electrochemical sensor of claim 1, wherein the coating of CNTs includes single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), or a combination of SWCNTs and MWCNTs.

9. The electrochemical sensor of claim 1, wherein the first electrode is operable as a working electrode, and the second electrode is operable as a counter electrode and/or a reference electrode in an electrochemical analysis technique.

10. The electrochemical sensor of claim 9, wherein the first electrode includes carbon, platinum, palladium, rhodium, iridium, nickel, or gold, and the second electrode is an Ag/AgCl electrode.

11. The electrochemical sensor of claim 1, wherein the electrode contingent includes a third electrode, and the first electrode is operable as a working electrode, and the second electrode is operable as a counter electrode, and the third electrode is operable as a reference electrode in an electrochemical analysis technique.

12. The electrochemical sensor of claim 11, wherein the first and the second electrodes include carbon, the third electrode is an Ag/AgCl electrode.

13. The electrochemical sensor of claim 1, wherein the enzymatic substance includes tyrosinase, catechol oxidase, polyphenol oxidase, or monophenol monooxygenase, or a combination thereof.

14. The electrochemical sensor of claim 1, wherein the conductive polymer film is cross-linked to the enzymatic substance using a solution containing bovine serum albumin, glutaraldehyde, and the enzymatic substance.

15. The electrochemical sensor of claim 1, further comprising a protective layer over the entrapment layer on the first electrode, wherein the protective layer includes a charge-exclusion material that allows charge neutral species and either positively or negatively charged species to pass through the protective layer.

16. The electrochemical sensor of claim 15, wherein the protective layer includes a Nafion film, wherein the protective layer prohibits negatively charged chemical species from passing through the protective layer.

17. The electrochemical sensor of claim 15, wherein the protective layer is formed over all of the electrodes of the electrode contingent.

18. The electrochemical sensor of claim 1, wherein the fluid sample includes a physiological fluid sample including blood, plasma, serum, saliva, or interstitial fluid samples.

19. The electrochemical sensor of claim 1, wherein the conductive polymer film includes poly(thionine), the enzymatic substance includes tyrosinase, and the target analyte includes L-DOPA in the fluid sample including whole blood, serum, or plasma, such that the electrochemical sensor is operable to detect the electrical signals associated with the concentration of the L-DOPA in the fluid sample when the electrochemical sensor is electrically coupled to an electrical circuit, by which the tyrosinase is capable to biocatalytically oxidize the L-DOPA to dopaquinone at the first electrode, such that the presence of dopaquinone causes the poly(thionine) film at the first electrode to oxidize, which concomitantly causes the dopaquinone to reduce back to L-DOPA.

20. A method to produce an analyte-selective electrochemical sensor, comprising:

providing a signal sensitivity-enhanced electrode on a substrate of an electrochemical sensor including carbon nanotubes (CNTs) on the surface of the electrode;

forming an entrapment layer over the signal sensitivity-enhanced electrode by depositing a solution of a polymer material on the surface of the electrode, and applying a fixed potential at the electrode and, subsequently, a continuously-cycled potential with respect to a reference electrode to produce a reversibly dopable conducting polymer film on the electrode; and

attaching an enzymatic substance to the entrapment layer by depositing a solution containing the enzymatic substance, an enzyme stabilizing agent, and a cross-linking agent on the conducting polymer film and drying the solution,

wherein the polymer material to form the conductive polymer film includes poly(thionine).

21. The method of claim 20, wherein the providing the signal sensitivity-enhanced electrode includes depositing a dispersed solution including the CNTs on the surface of a formed electrode on the substrate, and drying the solution to produce a layer of the CNTs.

22. The method of claim 20, wherein the providing the signal sensitivity-enhanced electrode includes forming a carbon-based screen printed electrode using a conductive ink material that contains the CNTs within the ink.

23. The method of claim 20, wherein the enzymatic substance is capable of catalyzing a target analyte of a fluid sample to cause a redox reaction detectable at the electrode when using an electrochemical analysis technique to operate the electrochemical sensor.

24. The method of claim 20, wherein the enzymatic substance includes tyrosinase, catechol oxidase, polyphenol oxidase, or monophenol monooxygenase, or a combination thereof.

25. The method of claim 20, wherein the enzyme stabilizing agent includes bovine serum albumin, and wherein the cross-linking agent includes glutaraldehyde.

26. The method of claim 20, further comprising:

producing a charge exclusion layer by depositing a solution containing Nafion and drying the solution.

27. The method of claim 26, wherein the depositing the charge-exclusion solution and drying is repeated multiple times.

28. The method of claim 20, wherein the electrochemical sensor is formed on a test strip.

29. The method of claim 20, further comprising storing the electrochemical sensor for future use.

30. A system to detect an analyte in a physiological sample, comprising:

a disposable electrochemical test strip sensor structured to include a substrate formed of an insulative material, an electrode contingent including a first electrode having a coating of carbon nanotubes (CNTs) and a second electrode on the substrate, an entrapment layer including a conductive polymer film on the first electrode to attach an enzymatic substance capable of catalyzing a target analyte of a physiological fluid sample, and first and second contact pads on the substrate that are respectively electrically coupled to the first and the second electrodes via electrically conductive conduits; and

a portable electrochemical analyzer including a housing including an opening to receive the disposable electrochemical test strip sensor, and an electrical circuit including bonding pads disposed in the opening to electrically couple to the electrochemical test strip sensor via first and the second contact pads when the electrochemical test strip sensor is inserted into the opening, wherein the electrical circuit is operable to supply an electrical waveform to the electrochemical test strip sensor to operate an electrochemical analysis technique and to detect electrical signals transduced by the first and second electrodes during the electrochemical analysis technique,

wherein the enzymatic substance is provided to cause a redox reaction involving the target analyte when the physiological fluid sample is deposited on the electrodes that produces charge carriers associated with the redox reaction capable of being electrically conducted across the conductive polymer film and detectable at the first electrode by the electrical circuit,

wherein the conductive polymer film includes poly(thionine).

31. The system of claim 30, wherein the portable electrochemical analyzer includes a signal conditioning circuit to amplify the detected electrical signals by the first electrode.

32. The system of claim 31, wherein the portable electrochemical analyzer includes a data processing unit including a processor to process data based on the detected electrical signal and a memory to store or buffer the data.

33. The system of claim 30, wherein the portable electrochemical analyzer includes a wireless communications unit to wirelessly transmit the detected electrical signal to an external computing device including a smartphone, a tablet, a pair of smartglasses, a smartwatch, a laptop computer, a desktop computer, or a network of computers in communication via the Internet.