POTENTIOMETRIC HYDROGEN PEROXIDE SENSOR

Abstract

Detection of hydrogen peroxide or phosphate related compounds in test or clinical samples by using potentiometric sensors wherein the reference electrode and a hydrogen peroxide selective electrode are both connected by a polyelectrolyte positioned in-between. More particularly a potentiometric sensor device for the detection of chemical species in solution with a novel configuration is disclosed.

Claims

1. In vitro use of a potentiometric cell to determine the concentration of hydrogen peroxide in an aqueous solution, wherein the potentiometric cell comprises: i. A reference electrode comprising a support which in turn comprises a conductive material; and ii. A hydrogen peroxide selective electrode comprising a porous redox sensitive surface consisting of platinum or gold, wherein each of the electrodes is connected to the other by a polyelectrolyte bridge provided between the electrodes, wherein the surfaces of each of the electrodes that contact the polyelectrolyte bridge provided between the electrodes permits to effectively close the potentiometric circuit; and wherein the use is characterized in that the aqueous solution is in direct contact with the working electrode, and in that instead of using the aqueous solution to close the potentiometric circuit between the two electrodes, the hydrogen peroxide selective electrode and the reference electrode (RE), the potentiometric cell uses the polyelectrolyte bridge to connect the electrodes.

2. The use according to claim 1, wherein the hydrogen peroxide selective electrode is positioned directly above the reference electrode.

3. The use according to any of claim 1 or 2, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum.

4. The use according to any of claims 1 to 3, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of perfluorosulfonic acid ionomers.

5. The use according to claim 4, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion or Aquivion, preferably Nafion.

6. The use according to claim 5, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion.

7. The use according to any of the precedent claims, wherein the reference electrode comprises a support which in turn comprises a conductive material selected from any of the following list consisting of: silver, platinum, gold, nickel, zinc, copper, aluminum and carbon.

8. The use according to any of the precedent claims, wherein the reference electrode and the hydrogen peroxide selective electrode are made of the same material, preferably gold or platinum.

9. The use according to any of the precedent claims, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum or gold and at least one layer of a proton exchange membrane on said redox sensitive surface, wherein said layer of a proton exchange membrane comprises a copolymer of Formula (II), ##STR00009## wherein x, y, m and n represent the numbers of repeat units, wherein m=0, n=1 and wherein the number of repeat units x and y are such that there are less than 15× units for each y.

10. The use according to claim 9, wherein said layer of a proton exchange membrane further comprises or conforms a mixture with a further PFSA copolymer selected from those of formula II wherein m=1 and n=1, or m=0 or 1 and n=1 to 5, or m=0 or 3 and n=2 to 5, or m=0 and n=2, and wherein x and y are such that there are less than 15× units for each y.

11. The use according to claim 9 or 10, wherein said layer of a proton exchange membrane further comprises a glucose oxidase entrapped therein or any oxidase with hydrogen peroxide as its product.

12. An in vitro method to determine the concentration of hydrogen peroxide in an aqueous solution, the method comprising; a. providing the aqueous solution in contact with the hydrogen peroxide selective electrode of a potentiometric sensor or cell comprising: i. A reference electrode comprising a support which in turn comprises a conductive material; and ii. A hydrogen peroxide selective electrode comprising a porous redox sensitive surface consisting of platinum or gold, wherein the hydrogen peroxide selective electrode is preferably positioned directly above the reference electrode, wherein each of the electrodes is connected to the other by a polyelectrolyte bridge provided between the electrodes, wherein the surfaces of each of the electrodes that contact the polyelectrolyte bridge provided between the electrodes permits to effectively close the potentiometric circuit; and wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of polyelectrolytes such as perfluorosulfonic acid ionomers or polyammonium ionomers; and b. measuring a potential difference between the reference electrode and the hydrogen peroxide selective electrode; and c. determining a concentration of hydrogen peroxide in the aqueous solution by evaluating the potential difference by using a voltmeter; wherein the method is characterized in that instead of using the aqueous solution to close the potentiometric circuit between the two electrodes, the hydrogen peroxide selective electrode and the reference electrode (RE), the potentiometric cell uses the polyelectrolyte bridge to connect the electrodes instead of the solution. The method according to claim 12, wherein the hydrogen peroxide selective electrode is positioned directly above the reference electrode.

13. The method according to claim 12, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum.

14. The method according to any of claims 12 to 13, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of perfluorosulfonic acid ionomers.

15. The method according to claim 14, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion or Aquivion, preferably Nafion.

16. The method according to claim 15, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion.

17. The method according to any of the precedent method claims, wherein the reference electrode comprises a support which in turn comprises a conductive material selected from any of the following list consisting of: silver, platinum, gold, nickel, zinc, copper, aluminum and carbon.

18. The method according to any of the precedent method claims, wherein the reference electrode and the hydrogen peroxide selective electrode are made of the same material, preferably gold or platinum.

19. The method according to any of the precedent method claims, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum or gold and at least one layer of a proton exchange membrane on said redox sensitive surface, wherein said layer of a proton exchange membrane comprises a copolymer of Formula (II), ##STR00010## wherein x, y, m and n represent the numbers of repeat units, wherein m=0, n=1 and wherein the number of repeat units x and y are such that there are less than 15× units for each y.

20. The method according to claim 20, wherein said layer of a proton exchange membrane further comprises or conforms a mixture with a further PFSA copolymer selected from those of formula II wherein m=1 and n=1, or m=0 or 1 and n=1 to 5, or m=0 or 3 and n=2 to 5, or m=0 and n=2, and wherein x and y are such that there are less than 15× units for each y.

21. The method according to claim 19 or 20, wherein said layer of a proton exchange membrane further comprises a glucose oxidase entrapped therein or any oxidase with hydrogen peroxide as its product.

22. A potentiometric cell to determine the concentration of hydrogen peroxide in an aqueous solution, wherein the potentiometric cell comprises: i. A reference electrode comprising a support which in turn comprises a conductive material; and ii. A hydrogen peroxide selective electrode comprising a porous redox sensitive surface consisting of platinum or gold, wherein each of the electrodes is connected to the other by a polyelectrolyte bridge provided between the electrodes, wherein the surfaces of each of the electrodes that contact the polyelectrolyte bridge provided between the electrodes permits to effectively close the potentiometric circuit, so that instead of using the aqueous solution to close the potentiometric circuit between the two electrodes, the hydrogen peroxide selective electrode and the reference electrode (RE), the potentiometric cell uses the polyelectrolyte bridge to connect the electrodes instead of the aqueous solution.

23. The potentiometric cell according to claim 22, wherein the potentiometric cell is adapted so that the aqueous solution to be tested is only in contact with the hydrogen peroxide selective electrode and does not close the potentiometric circuit between the two electrodes.

24. The potentiometric cell according to claim 22 or 23, wherein the hydrogen peroxide selective electrode is positioned directly above the reference electrode.

25. The potentiometric cell according to any of claims 22 to 24, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum.

26. The potentiometric cell according to any of claims 22 to 25, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of perfluorosulfonic acid ionomers.

27. The potentiometric cell according to claim 26, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion or Aquivion, preferably Nafion.

28. The potentiometric cell according to claim 27, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion.

29. The potentiometric cell according to any of claims 22 to 28, wherein the reference electrode comprises a support which in turn comprises a conductive material selected from any of the following list consisting of: silver, platinum, gold, nickel, zinc, copper, aluminum and carbon.

30. The potentiometric cell according to any of claims 22 to 29, wherein the reference electrode and the hydrogen peroxide selective electrode are made of the same material, preferably gold or platinum.

31. The potentiometric cell according to any of claims 22 to 30, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum or gold and at least one layer of a proton exchange membrane on said redox sensitive surface, wherein said layer of a proton exchange membrane comprises a copolymer of Formula (II), ##STR00011## wherein x, y, m and n represent the numbers of repeat units, wherein m=0, n=1 and wherein the number of repeat units x and y are such that there are less than 15× units for each y.

32. The potentiometric cell according to claim 31, wherein said layer of a proton exchange membrane further comprises or conforms a mixture with a further PFSA copolymer selected from those of formula II wherein m=1 and n=1, or m=0 or 1 and n=1 to 5, or m=0 or 3 and n=2 to 5, or m=0 and n=2, and wherein x and y are such that there are less than 15× units for each y.

33. The potentiometric cell according to claim 31 or 32, wherein said layer of a proton exchange membrane further comprises a glucose oxidase entrapped therein or any oxidase with hydrogen peroxide as its product.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0074] FIG. 1: Paper-based sensor configuration for hydrogen peroxide detection (right) and for glucose detection (left) a. Nafion® 5% membrane, b. Pt sputtered paper strip, c. Nafion® 10% d. Conductive paper strip, e. Plastic mask. f. GOx enzyme. In the left are the components of the sensor, and in the right the assembled sensor.

[0075] FIG. 2: Response time of a sensor to concentration of hydrogen peroxide (logarithmic units) in PBS: left) time 2500 to 4000 s refers to a sensor in a cell of 5 mL volume and right) time 4500 to 6500 s refers to a single drop of 50 μL volume.

[0076] FIG. 3: calibration curve of a sensor (corresponding to data from FIG. 2) blue data obtained in solution (PBS 5 mL) and in drop (PBS 50 uL)

[0077] FIG. 4: a) Time trace of glucose sensor and b) corresponding calibration curve in PBS (5 mL).

[0078] FIG. 5: Traditional approach and for the detection of chemical species in solution by using potentiometric sensors.

[0079] FIG. 6. A schematic view of such proposed configuration.

[0080] The following examples merely illustrate the present invention and do not limit the same.

Examples

Materials and Methods

[0081] In this invention, a new cell geometry using metal-sputtered papers in working and reference electrodes, and a Nafion membrane as a conductive media, is applied in the construction of a paper-based potentiometric all-solid-state sensor for hydrogen peroxide detection, i.e. the biomarker of the oxidase enzyme reaction as well as, for glucose as a model biomarker.

Experimental Section

[0082] Reagents. Nafion® 117 solution (10% in a mixture of lower aliphatic alcohols and water); glucose oxidase (GOx) (from Aspergillus niger type X-S, lyophilized powder (100,000-250.000 units/g) D-glucose); hydrogen peroxide (30 wt. % in water) and D-Glucose (Glu) were purchased from Sigma-Aldrich. Phosphate buffer saline (PBS) pH=7.4 (0.1 M NaCl, 0.003 M KCl, 0.1 M Na2HPO4, 0.02 M K2HPO4) were prepared using 18.2 MΩ cm-1 double deionized water (Milli-Q water systems, Merck Millipore).

[0083] Sputtered platinum paper. Whatman® Grade 5 qualitative filter paper circles were coated with Pt using a radiofrequency sputtering process (ATC Orion 8-HV, AJA International) operated at 3 mTorr, for 65 s at 200 W.

[0084] Paper sensor construction (FIG. 1). two conductive paper strips are used, namely: the upper strip (made with a Pt sputtered paper) acts as WE (working electrode); the lower strip is a conducting paper that acts as reference electrode (RE). WE and RE are glued using a drop of Nafion® 10% (sandwiched between the plastic masks). Finally, a drop of Nafion® 5% is located over the electrochemically active window, covering the exposed area of the WE. In all cases the conductive paper strips were cut with a width of 0.4 cm.

Results

Hydrogen Peroxide Detection in Solution and in Drop

[0085] FIG. 2 shows the response and calibration curve for the novel sensors to the addition of H.sub.2O.sub.2 in PBS (5 mL cell). The bridge indeed connects the working to the reference electrode (Nafion solution). The additions give a negative response (from −7 to −1) with a sensitivity of −90 mV per decade in the linear range from −5 to −3 (FIG. 3 reports the corresponding calibration curve). Importantly, these results confirm that the proposed potentiometric cell—using Nafion to connect WE and RE instead of the solution—effectively closes the circuit and allows detecting H.sub.2O.sub.2 in solution. To the best of our knowledge, this is the first report for this kind of configuration for the detection of chemical species in solution.

[0086] Moreover, the novel configuration allows detection in reduced volume such as shown in FIG. 2 right. The detection was performed in a single drop of 50 μL reducing the volume 2 order of magnitude. Noteworthy further reduction of volume down to the single μL is possible based on the geometry optimization. Analytical figures are comparable to the ones in solution although with a shifted linear range. The sensitivity here was 100 mV/dec in the −4 −2 linear range. Furthermore, the linear range corresponds to the clinical range of blood glucose and it is therefore of utmost importance.

Glucose Detection

[0087] In addition, a glucose sensor was constructed: GOx enzyme was entrapped in the first layer of Nafion so that it catalyzes the oxidation of glucose added to the solution producing H2O2, which is detected by the working electrode. FIG. 4a. shows the time trace obtained in artificial serum (AS). As expected, the measured EMF decreases with the increasing concentration of glucose (sensitivity of −53 mV/dec in the linear range −3.5 −2.5). Thus, this result confirms that the new configuration could be used for real application having an analytical performance similar to other reported glucose biosensors.

CLAUSES

[0088] 1. A potentiometric cell suitable for selectively measuring hydrogen peroxide in solution, which comprises: [0089] a. A reference electrode comprising a support which in turn comprises a conductive material; and [0090] b. A hydrogen peroxide selective electrode comprising a porous redox sensitive surface consisting of platinum or gold,
wherein the hydrogen peroxide selective electrode is positioned directly above the reference electrode, wherein each of the electrodes is connected to the other by a polyelectrolyte bridge provided between the electrodes, and wherein the surfaces of each of the electrodes that contact the polyelectrolyte bridge provided between the electrodes permits to effectively close the circuit allowing the detection of the chemical species in solution; and
wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of polyelectrolytes such as perfluorosulfonic acid ionomers or polyammonium ionomers. [0091] 2. The potentiometric cell of clause 1, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum. [0092] 3. The potentiometric cell of clause 1 or 2, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of perfluorosulfonic acid ionomers. [0093] 4. The potentiometric cell of clause 3, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion or Aquivion, preferably Nafion. [0094] 5. The potentiometric cell of clause 3, wherein the polyelectrolyte bridge connecting the electrodes comprises or is made of Nafion. [0095] 6. The potentiometric cell of any of the previous clauses, wherein the reference electrode comprises a support which in turn comprises a conductive material selected from any of the following list consisting of: silver, platinum, gold, nickel, zinc, copper, aluminum and carbon. [0096] 7. The potentiometric cell according to the any of the precedent clauses, wherein the hydrogen peroxide selective electrode comprises a porous redox sensitive surface consisting of platinum or gold and at least one layer of a proton exchange membrane on said redox sensitive surface, wherein said layer of a proton exchange membrane comprises a copolymer of Formula (II),

##STR00008##  wherein x, y, m and n represent the numbers of repeat units, wherein m=0, n=1 and wherein the number of repeat units x and y are such that there are less than 15× units for each y. [0097] 8. The potentiometric cell according to clause 7, wherein said layer of a proton exchange membrane further comprises or conforms a mixture with a further PFSA copolymer selected from those of formula II wherein m=1 and n=1, or m=0 or 1 and n=1 to 5, or m=0 or 3 and n=2 to 5, or m=0 and n=2, and wherein x and y are such that there are less than 15× units for each y. [0098] 9. The potentiometric cell according to clause 7 or 8, wherein said layer of a proton exchange membrane further comprises a glucose oxidase entrapped therein or any oxidase with hydrogen peroxide as its product. [0099] 10. In vitro use of the potentiometric cell of any of the precedent clauses, to selectively and directly determine the concentration of hydrogen peroxide in an aqueous solution. [0100] 11. The in vitro use according to clause 10, wherein said aqueous solution is a biological fluid such as whole blood, preferably undiluted whole blood, intracellular fluids, saliva, cerebrospinal fluid, blood sera, blood plasma, sweat and urine.